Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in duckweed plants

ABSTRACT

Methods for altering the N-glycosylation pattern of proteins in higher plants are provided. In some embodiments, the methods comprise introducing into a duckweed plant a recombinant RNAi construct that provides for the inhibition of expression of α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT). Use of these RNAi constructs to inhibit or suppress expression of both of these enzymes, and isoforms thereof, advantageously provides for the production of endogenous and heterologous proteins having a “humanized” N-glycosylation pattern without impacting plant growth and development. Stably transformed higher plants, including duckweed plants, having this protein N-glycosylation pattern are provided. Glycoprotein compositions, including monoclonal antibody compositions, having substantially homogeneous glycosylation profiles, and which are substantially homogeneous for the G0 glycoform, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/759,298, filed Jan. 17, 2006; U.S. Provisional Application No.60/790,373, filed Apr. 7, 2006; U.S. Provisional Application No.60/791,178, filed Apr. 11, 2006; U.S. Provisional Application No.60/812,702, filed Jun. 9, 2006; U.S. Provisional Application No.60/836,998, filed Aug. 11, 2006; and U.S. Provisional Application No.60/860,358, filed Nov. 21, 2006; the contents of each of which arehereby incorporated herein in their entirety by reference.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

A joint research agreement was executed on Mar. 4, 2005, and Mar. 10,2005, for ddRNAi technology in cells, tissues, or whole plants of thefamily Lemnaceae. The names of the parties executing the joint researchagreement are Biolex, Inc., now Biolex Therapeutics, Inc., having itsregistered address at 158 Credle Street, Pittsboro, N.C. 27312 USA, andCommonwealth Scientific and Industrial Research Organisation ABN 41 687119 230, a body corporate established under the Science and IndustryResearch Act 1949, of Limestone Avenue, Campbell in the AustralianCapital Territory, Australia.

FIELD OF THE INVENTION

The present invention is related to the field of plant molecularbiology, more particularly to the field of recombinant mammalian proteinproduction in plants.

BACKGROUND OF THE INVENTION

A number of plant species have been targeted for use in “molecularfarming” of mammalian proteins of pharmaceutical interest. These plantexpression systems provide for low cost production of biologicallyactive mammalian proteins and are readily amenable to rapid andeconomical scale-up (Ma et al. (2003) Nat. Rev. Genet. 4:794-805; Raskinet al. (2002) Trends Biotechnol. 20:522-531). Large numbers of mammalianand plant proteins require post-translational processing for properfolding, assembly, and function. Of these modifications, the differencesin glycosylation patterns between plants and mammals offer a challengeto the feasibility of plant expression systems to produce high qualityrecombinant mammalian proteins for pharmaceutical use.

As peptides move through the endoplasmic reticulum (ER) and Golgisubcellular compartments, sugar residue chains, or glycans, areattached, ultimately leading to the formation of glycoproteins. Thelinkage between the sugar chains and the peptide occurs by formation ofa chemical bond to only one of four protein amino acids: asparagine,serine, threonine, and hydroxlysine. Based on this linkage pattern, twobasic types of sugar residue chains in glycoproteins have beenrecognized: the N-glycoside-linked sugar chain (also referred to asN-linked glycan or N-glycan), which binds to asparagine residues on thepeptide; and the O-glycoside-linked sugar chain, which binds to serine,threonine, and hydroxylysine residues on the peptide.

The N-glycoside-linked sugar chains, or N-glycans, have variousstructures (see, for example, Takahashi, ed. (1989) BiochemicalExperimentation Method 23—Method for Studying Glycoprotein Sugar Chain(Gakujutsu Shuppan Center), but share a common oligomannosidic core (seeFIG. 29A). The initial steps in the glycosylation pathway leading to theformation of N-glycans are conserved in plants and animals. However, thefinal steps involved in complex N-glycan formation differ (Lerouge etal. (1998) Plant Mo. Biol. 38:31-48; Steinkellner and Strasser (2003)Ann. Plant Rev. 9:181-192). Plants produce glycoproteins with complexN-glycans having a core bearing two N-acetylglucosamine (GlcNAc)residues that is similar to that observed in mammals. However, in plantglycoproteins this core is substituted by a β1,2-linked xylose residue(core xylose), which residue does not occur in humans, Lewis^(a)epitopes, and an α1,3-linked fucose (core α[1,3]-fucose) instead of anα1,6-linked core fucose as in mammals (see, for example, Lerouge et al.(1998) Plant Mol. Biol. 38:31-48 for a review) (see also FIG. 29B). Boththe α(1,3)-fucose and β(1,2)-xylose residues reportedly are, at leastpartly, responsible for the immunogenicity of plant glycoproteins inmammals (see, for example, Ree et al. (2000) J. Biol. Chem.15:11451-11458; Bardor et al. (2003) Glycobiol. 13:427-434;Garcia-Casado et al. (1996) Glycobiol. 6:471-477). Therefore removal ofthese potentially allergenic sugar residues from mammalian glycoproteinsrecombinantly produced in plants would overcome concerns about the useof these proteins as pharmaceuticals for treatment of humans.

A number of recombinantly produced glycoproteins currently serve astherapeutics or are under clinical investigation. Examples include theinterferons (IFNs), erythropoietin (EPO), tissue plasminogen activator(tPA), antithrombin, granulocyte-macrophage colony stimulating factor(GM-CSF), and therapeutic monoclonal antibodies (mABs). Theoligosaccharide component of the N-glycan structures of glycoproteinscan influence their therapeutic efficacy, as well as their physicalstability, resistance to protease attack, pharmacokinetics, interactionwith the immune system, and specific biological activity. See, forexample, Jenkins et al. (1996) Nature Biotechnol. 14:975-981.

Methods are needed to alter the glycosylation pattern in plantexpression systems, specifically to inhibit plant-specific glycosylationof the eukaryotic core structure, to advantageously produce recombinantmammalian proteins with a humanized glycosylation pattern.

BRIEF SUMMARY OF THE INVENTION

Methods for altering the N-glycosylation pattern of proteins in higherplants are provided. The methods comprise stably transforming the plantwith at least one recombinant nucleotide construct that provides for theinhibition of expression of α1,3-fucosyltransferase (FucT) andβ1,2-xylosyltransferase (XylT) in a plant. Use of these constructs toinhibit or suppress expression of one or both of these enzymes, andisoforms thereof, advantageously provides for the production ofendogenous and heterologous proteins having a “humanized”N-glycosylation pattern without impacting plant growth and development.Stably transformed higher plants having this protein N-glycosylationpattern are provided. In some embodiments, the plant is a crop plantthat is a member of the dicots, such as pea, alfalfa, and tobacco; inother embodiments, the plant is a crop plant that is a monocot, such asrice or maize. In yet other embodiments, the plant is a member of theLemnaceae family, for example, a Lemna sp.

The transgenic plants of the invention have the ability to producerecombinant mammalian proteins having an N-glycosylation pattern that ismore similar to that of the mammalian host. Thus, in some embodiments,the recombinantly produced mammalian proteins are glycoproteinscomprising complex N-glycans having a reduction in the attachment of theplant-specific α(1,3)-fucose and β(1,2)-xylose residues. In otherembodiments, these recombinantly produced glycoproteins comprise complexN-glycans that are devoid of these plant-specific residues. In yet otherembodiments, these recombinantly produced glycoproteins haveGlcNAc₂Man₃GlcNAc₂ as the single glycan species attached to theasparagine glycosylation site(s) within the glycoprotein.

In some embodiments, the recombinantly produced glycoprotein is amonoclonal antibody. In this manner, the present invention providestransgenic plants that are capable of producing glycan-optimizedmonoclonal antibodies that specifically bind a target protein ofinterest, including monoclonal antibodies having increased effectorfunction. Thus, in some embodiments, the recombinantly producedglycan-optimized monoclonal antibodies comprise complex N-glycans thathave a reduction in the attachment of α(1,3)-linked fucose residues,thereby increasing ADCC activity of these antibodies. In otherembodiments, the recombinantly produced monoclonal antibodies comprisecomplex N-linked glycans that are devoid of these plant-specific fucoseresidues. In this manner, the present invention provides for theproduction of a monoclonal antibody composition, wherein at least 90% ormore of the intact antibody is represented by a single glycoform, moreparticularly, the G0 glycoform. Thus, in some embodiments of theinvention, the recombinantly produced monoclonal antibodies haveincreased effector function, wherein the ADCC activity is increasedand/or the ratio of ADCC/CDC activity is increased. In some of theseembodiments, the recombinantly produced monoclonal antibodies havedecreased CDC activity, which can advantageously reduce the potentialfor adverse side effects related to CDC activation upon administration.These glycan-optimized monoclonal antibodies advantageously can be usedto alter current routes of administration and current therapeuticregimens, as their increased effector function means they can be dosedat lower concentrations and with less frequency, thereby reducing thepotential for antibody toxicity and/or development of antibodytolerance. Furthermore, their improved effector function yields newapproaches to treating clinical indications that have been previouslybeen resistant or refractory to treatment with the correspondingmonoclonal antibody produced in other recombinant host systems.

Compositions for practicing the methods of the invention are provided.The compositions comprise novel isolated polynucleotides andpolypeptides encoding a Lemna minor α1,3-fucosyltransferase andβ1,2-xylosyltransferase, and variants and fragments thereof. Recombinantnucleotide constructs that target expression of these two proteins, orexpression of variants thereof, are also provided, as are plant cells,plant tissues, plants, and seeds comprising these recombinantconstructs.

BRIEF DESCRIPTION OF THE FORMAL DRAWINGS

FIG. 1 sets forth the DNA (SEQ ID NO:1; coding sequence set forth in SEQID NO:2) and amino acid (SEQ ID NO:3) sequences for the Lemna minorα1,3-fucosyltransferase (FucT). The coding sequence is shown in bold.Nucleotides denoted by the single underline (-) correspond to the FucTforward fragment within the RNAi expression cassette designed to inhibitexpression of FucT (see FIG. 5); nucleotides denoted by the doubleunderline (=) correspond to the spacer sequence within this RNAiexpression cassette. The FucT reverse fragment of the RNAi expressioncassette is the antisense of the FucT forward fragment shown here.

FIG. 2 sets forth an alignment of the Lemna minor FucT of SEQ ID NO:3with α1,3-fucosyltransferases from Triticum aestivum (set forth in SEQID NO: 30), Hordeum vulgare (set forth in SEQ ID NO: 31), Oryza sativa(set forth in SEQ ID NO: 32), Medicago sativa (set forth in SEQ ID NO:33), and Arabidopsis thaliana (set forth in SEQ ID NO: 34). Theconsensus sequence of the six sequences is set forth in SEQ ID NO: 35.

FIG. 3 sets forth the DNA (SEQ ID NO:4; coding sequence set forth in SEQID NO:5) sequence for the Lemna minor β1,2-xylosyltransferase (XylT)isoform #1 and the encoded amino acid (SEQ ID NO:6) sequence.Nucleotides denoted by the single underline (-) correspond to the XylTforward fragment within the RNAi expression cassette designed to inhibitexpression of XylT (see FIG. 6); nucleotides denoted by the doubleunderline (=) correspond to the spacer sequence within this RNAiexpression cassette. The XylT reverse fragment of the RNAi expressioncassette is the antisense of the XylT forward fragment shown here.

FIG. 4 sets forth an alignment of the Lemna minor XylT of SEQ ID NO:6with β1,2-xylosyltransferases from Medicago sativa (set forth in SEQ IDNO: 36), Oryza sativa (set forth in SEQ ID NO: 37), Arabidopsis thaliana(set forth in SEQ ID NO: 38), Saccharum officinarum (set forth in SEQ IDNO: 39), and Nicotiana tabacum (set forth in SEQ ID NO: 40). Theconsensus sequence of the six sequences is set forth in SEQ ID NO: 41.

FIG. 5 sets forth one strategy for designing a single-gene RNAi knockoutof Lemna minor FucT.

FIG. 6 sets forth one strategy for designing a single-gene RNAi knockoutof Lemna minor XylT based on the DNA sequence for XylT isoform #1.

FIG. 7 sets forth one strategy for designing a double-gene RNAi knockoutof Lemna minor FucT and XylT where the XylT portion of the RNAi knockoutis based on the DNA sequence for XylT isoform #1.

FIG. 8 shows the Fuc02 construct comprising an RNAi expression cassettedesigned for single-gene RNAi knockout of Lemna minor FucT. Expressionof the FucT inhibitory sequence (denoted by FucT forward and FucTreverse arrows; see FIG. 5) is driven by an operably linked expressioncontrol element (denoted as AocsAocsAocsAmasPmas) comprising threeupstream activating sequences (Aocs) derived from the Agrobacteriumtumefaciens octopine synthase gene operably linked to a promoter derivedfrom an Agrobacterium tumefaciens mannopine synthase gene (AmasPmas).RbcS leader, rubisco small subunit leader sequence; ADH1, intron ofmaize alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium tumefaciansnopaline synthetase (nos) terminator sequence.

FIG. 9 shows the Xyl02 construct comprising an RNAi expression cassettedesigned for single-gene RNAi knockout of Lemna minor XylT. Expressionof the XylT inhibitory sequence (denoted by XylT forward and XylTreverse arrows; see FIG. 6) is driven by the operably linkedAocsAocsAocsAmasPmas expression control element. RbcS leader, rubiscosmall subunit leader sequence; ADH1, intron of maize alcoholdehydrogenase 1 gene; nos-ter, Agrobacterium tumefacians nopalinesynthetase (nos) terminator sequence.

FIG. 10 shows the XF02 construct comprising a chimeric RNAi expressioncassette designed for double-gene RNAi knockout of Lemna minorFucT/XylT. The hairpin RNA is expressed as a chimeric sequence (achimeric hairpin RNA), where fragments of the two genes are fusedtogether and expressed as one transcript. Expression of the FucT/XylTinhibitory sequence (denoted by FucT and XylT forward arrows and XylTand FucT reverse arrows; see FIG. 7) is driven by the operably linkedAocsAocsAocsAmasPmas expression control element. RbcS leader, rubiscosmall subunit leader sequence; ADH1, intron of maize alcoholdehydrogenase 1 gene; nos-ter, Agrobacterium tumefacians nopalinesynthetase (nos) terminator sequence.

FIG. 11 shows the XF03 construct comprising an RNAi expression cassettedesigned for double-gene RNAi knockout of Lemna minor FucT/XylT. Thecassette expresses two RNAi hairpins, one targeting expression of FucT,the other targeting expression of XylT. Expression of the FucTinhibitory sequence (denoted by FucT forward and FucT reverse arrows;see FIG. 5) is driven by an operably linked expression control elementcomprising the Lemna minor ubiquitin promoter plus 5′ UTR (LmUbqpromoter) and intron (LmUbq intron) (see SEQ ID NO:7). Expression of theXylT inhibitory sequence (denoted by XylT forward and XylT reversearrows; see FIG. 6) is driven by the operably linkedAocsAocsAocsAmasPmas expression control element. RbcS leader, rubiscosmall subunit leader sequence; ADH1, intron of maize alcoholdehydrogenase 1 gene; nos-ter, Agrobacterium tumefacians nopalinesynthetase (nos) terminator sequence.

FIG. 12 shows the mAbI04 construct that provides for co-expression of anIgG1 monoclonal antibody (referred to herein as mAbI) and thedouble-gene knockout of Lemna minor FucT and XylT, wherein a chimerichairpin RNA targeting expression of the FucT and XylT is expressed.Expression of the FucT/XylT inhibitory sequence (denoted by FucT andXylT forward arrows and XylT and FucT reverse arrows; see FIG. 7) isdriven by an operably linked expression control element comprising theSpirodella polyrrhiza ubiquitin promoter plus 5′ UTR (SpUbq promoter)and intron (SpUbq intron) (see SEQ ID NO: 8). Expression of the IgG1light chain is driven by an operably linked expression control elementcomprising the L. minor ubiquitin promoter plus 5′ UTR (LmUbq promoter)and intron (LmUbq intron). Expression of the IgG1 heavy chain is drivenby the operably linked AocsAocsAocsAmasPmas expression control element.RbcS leader, rubisco small subunit leader sequence; ADH1, intron ofmaize alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium tumefaciansnopaline synthetase (nos) terminator sequence.

FIG. 13 shows the mAbI05 construct that provides for co-expression ofmAbI and the double-knockout of Lemna minor FucT and XylT, wherein twohairpin RNAs are expressed, one targeting expression of FucT, the othertargeting expression of the XylT. Expression of the FucT inhibitorysequence (denoted by FucT forward and FucT reverse arrows; see FIG. 5)is driven by an operably linked expression control element comprisingthe S. polyrrhiza ubiquitin promoter plus 5′ UTR (SpUbq promoter) andintron (SpUbq intron). Expression of the XylT inhibitory sequence(denoted by XylT forward and XylT reverse arrows; see FIG. 6) is drivenby an operably linked expression control element comprising the Lemnaaequinoctialis ubiquitin promoter plus 5′ UTR (LaUbq promoter) andintron (LaUbq intron) (see SEQ ID NO:9). Expression of the IgG1 lightchain is driven by an operably linked expression control elementcomprising the L. minor ubiquitin promoter plus 5′ UTR (LmUbq promoter)and intron (LmUbq intron). Expression of the IgG1 heavy chain is drivenby the operably linked AocsAocsAocsAmasPmas expression control element.RbcS leader, rubisco small subunit leader sequence; ADH1, intron ofmaize alcohol dehydrogenase 1 gene; nos-ter, Agrobacterium tumefaciansnopaline synthetase (nos) terminator sequence.

FIG. 14 shows the mAbI01 construct that provides for expression of mAbI,where FucT and XylT expression are not suppressed. Expression of theIgG1 light chain and IgG1 heavy chain are independently driven by theoperably linked AocsAocsAocsAmasPmas expression control element. RbcSleader, rubisco small subunit leader sequence; ADH1, intron of maizealcohol dehydrogenase 1 gene; nos-ter, Agrobacterium tumefaciansnopaline synthetase (nos) terminator sequence. mAbI01 is referred to asthe “wild-type” mAbI01 construct as the expressed mAbI exhibits theglycosylation profile of wild-type L. minor.

FIGS. 15 and 16 show the primary screening data for transgenic RNAi L.minor plant lines comprising the XF02 construct of FIG. 10.

FIG. 17 shows primary screening data for transgenic RNAi L. minor plantlines comprising the mAbI04 construct of FIG. 12 and mAbI05 construct ofFIG. 13.

FIG. 18 shows the structure and molecular weight of derivatizedwild-type L. minor mAb N-glycans. “GnGn” represents theGlcNAc₂Man₃GlcNAc₂ N-glycan species, also referred to as a G0 N-glycanspecies. “GnGnX” represents the GlcNAc₂Man₃GlcNAc₂ N-glycan species withthe plant-specific β(1,2)-xylose residue attached. “GnGnXF” representsthe GlcNAc₂Man₃GlcNAc₂ N-glycan species with the plant-specificβ(1,2)-xylose residue and plant-specific α(1,3)-fucose residue attached.

FIG. 19 shows that the wild-type mAbI01 construct (shown in FIG. 15)providing for expression of the mAbI monoclonal IgG1 antibody in L.minor, without RNAi suppression of L. minor FucT and XylT, produces anN-glycosylation profile with three major N-glycan species, including onespecies having the β1,2-linked xylose and one species having both theβ1,2-linked xylose and core α1,3-linked fucose residues.

FIG. 20 shows that the N-glycosylation profile in FIG. 19 is confirmedwith liquid chromatography mass spectrometry (LC-MS).

FIG. 21 shows that the N-glycosylation profile in FIG. 19 is confirmedwith MALDI analysis.

FIG. 22 shows an overlay of the relative amounts of the various N-glycanspecies of mAbI produced in the wild-type L. minor line comprising themAbI01 construct (no suppression of FucT or XylT) and in the twotransgenic L. minor lines comprising the mAbI04 construct of FIG. 12(providing for coexpression of mAbI and the chimeric RNAi constructtargeting both L. minor FucT and XlT). Note the enrichment of the GnGn(i.e., G0) glycan species, with no β1,2-linked xylose or coreα1,3-linked fucose residues attached, and the absence of the specieshaving the β1,2-linked xylose or both the β1,2-linked xylose and coreα1,3-linked fucose residues. “MGn” represents an N-glycan precursor,wherein the trimannose core structure, Man₃GlcNAc₂, has oneN-acetylglucosamine attached to the 1,3 mannose arm. “GnM” represents anN-glycan precursor, wherein the trimannose core structure, Man₃GlcNAc₂,has one N-acetylglucosamine attached to the 1,6 mannose arm. TheseN-glycan precursors represent a trace amount of the total N-glycanspresent in the sample.

FIG. 23 shows that the profile in FIG. 22 is confirmed with mass spec(LC-MS).

FIG. 24 shows that the profile in FIG. 22 is confirmed with MALDIanalysis.

FIG. 25 shows intact mass analysis of mAbI compositions produced inwild-type L. minor (line 20) comprising the mAbI01 construct. When XylTand FucT expression are not suppressed in L. minor, the recombinantlyproduced mAbI composition is heterogeneous, comprising at least 9different glycoforms, with the G0XF³ glycoform being the predominatespecies present. Note the very minor peak representing the G0 glycoform.

FIG. 26 shows intact mass analysis of mAbI compositions produced intransgenic L. minor (line 15) comprising the mAbI04 construct of FIG.12. When XylT and FucT expression are suppressed in L. minor using thischimeric RNAi construct, the intact mAbI composition is substantiallyhomogeneous for G0 N-glycans, with only trace amounts of precursorN-glycans present (represented by the GnM and MGn precursor glycanspecies). In addition, the mAbI composition is substantially homogeneousfor the G0 glycoform, wherein both glycosylation sites are occupied bythe G0 N-glycan species, with three minor peaks reflecting trace amountsof precursor glycoforms (one peak showing mAbI having an Fc regionwherein the C_(H)2 domain of one heavy chain has a G0 glycan speciesattached to Asn 297, and the C_(H)2 domain of the other heavy chain isunglycosylated; another peak showing mAbI having an Fc region whereinthe C_(H)2 domain of one heavy chain has a G0 glycan species attached toAsn 297, and the C_(H)2 domain of the other heavy chain has the GnM orMGn precursor glycan attached to Asn 297; and another peak showing mAbIhaving an Fc region wherein the Asn 297 glycosylation site on each ofthe C_(H)2 domains has a G0 glycan species attached, with a third G0glycan species attached to an additional glycosylation site within themAbI structure).

FIG. 27 shows intact mass analysis of the mAbI compositions produced intransgenic L. minor (line 72) comprising the mAbI05 construct of FIG.13. When XylT and FucT expression are suppressed in L. minor using thisconstruct, the intact mAbI composition is substantially homogeneous forG0 N-glycans, with only trace amounts of precursor N-glycan speciespresent (represented by the GnM and MGn precursor glycan species). Inaddition, the mAbI composition is substantially homogeneous (at least90%) for the G0 glycoform, with the same three minor peaks reflectingprecursor glycoforms as obtained with the mAbI04 construct.

FIG. 28 summarizes two possible designs for targeting expression ofindividual FucT and XylT genes.

FIG. 29A shows the common oligomannosidic core structure of complexN-glycans of glycoproteins produced in plants and animals. In mammals,the core structure can include a fucose residue in which 1-position ofthe fucose is bound to 6-position of the N-acetylgucosamine in thereducing end through an α bond (i.e., α(1,6)-linked fucose). FIG. 29Bshows the plant-specific modifications to these N-glycans. The mammalianR groups can be one of the following: (a) R=GlcNAcβ(1,2); (b)R=Galβ(1,4)-GlcNAcβ(1,2); (c) R=NeuAcα(2,3)-Galβ(1,4)-GlcNAcβ(1,2); (d)R=NeuGcα(2,3)-Galβ(1,4)-GlcNAcβ(1,2); and (e)R=Galα(1,3)-Galβ(1,4)-GlcNAcβ(1,2). The plant R groups can be one of thefollowing: (a) R=null; (b) R=GlcNAcβ(1,2);

Abbreviations: Man, mannose; GlcNAc, N-acetylglucosamine; Xyl, xylose;Fuc, fucose; Gal, galactose; NeuAc (neuraminic acid (sialic acid); *,reducing end of sugar chain that binds to asparagine

FIG. 30 shows the G0, G0X, and G0XF³ species of N-linked glycans ofglycoproteins referred to in the description and claims of the presentinvention, along with the alternate nomenclature used herein.

FIG. 31 sets forth the partial cDNA (SEQ ID NO:19; coding sequence setforth in SEQ ID NO:20) sequence for the Lemna minorβ1,2-xylosyltransferase (XylT) isoform #2, and partial amino acid (SEQID NO:21) sequence encoded thereby. Nucleotides denoted by the singleunderline (-) correspond to the XylT forward fragment within the RNAiexpression cassette designed to inhibit expression of XylT (see FIG.33); nucleotides denoted by the double underline (=) correspond to thespacer sequence within this RNAi expression cassette. The XylT reversefragment of the RNAi expression cassette is the antisense of the XylTforward fragment shown here.

FIG. 32 sets forth an alignment of the Lemna minor XylT isoform #1 ofSEQ ID NO:6 with the Lemna minor partial-length XylT isoform #2 of SEQID NO:21.

FIG. 33 sets forth one strategy for designing a single-gene RNAiknockout of Lemna minor XylT based on the partial DNA sequence for XylTisoform #2.

FIG. 34 sets forth one strategy for designing a double-gene RNAiknockout of Lemna minor FucT and XylT, where the XylT portion of theRNAi knockout is based on the partial DNA sequence for XylT isoform #2.

FIG. 35 shows receptor binding activity of the CHO-derived andSP2/0-derived mAbI product for the FcγRIIIa on freshly isolated human NKcells.

FIG. 36 shows receptor binding activity of the wild-type Lemna-derivedmAbI product and the transgenic Lemna-derived mAbI product for theFcγRIIIa on freshly isolated human NK cells collected from Donor 1.

FIG. 37 shows receptor binding activity of the wild-type Lemna-derivedmAbI product and the transgenic Lemna-derived mAbI product for theFcγRIIIa on freshly isolated human NK cells collected from Donors 2 and3.

FIG. 38 shows receptor binding activity of the Sp2/0-derived mAbIproduct, the wild-type Lemna-derived mAbI product, and the transgenicLemna-derived mAbI product for the mouse FcγRIV.

FIG. 39 shows a diagram of the MDXA04 binary expression vector for RNAisilencing of FucT and XylT activity in Lemna. Hatched regions show theposition of the heavy (H) and light (L) chain variable region genesequences of fully human mAb1 kappa antibody MDX-060 and the chimerichairpin RNA (RNAi) designed to target silencing of endogenous Lemnagenes encoding FucT and XylT. Promoters: P1, P2, and P3; terminator: T;selectable marker: SM; left border: LB; right border, RB. The MDXA01expression vector used to express the MDX-060 mAb in wild-type Lemna didnot contain the hairpin RNA region.

FIG. 40 shows glycosyltransferase activity in Lemna wild-type andMDX-060 LEX^(Opt) RNAi lines. Microsomal membranes from wild-type (WT)and MDX-060 LEX^(Opt) RNAi (line numbers are indicated) plants wereincubated in the presence of a reaction buffer containing GDP-Fuc,UDP-Xyl and GnGn-dabsyl-peptide acceptor. Mass peaks corresponding tofucosylated (white bars) or xylosylated (black bars) productssynthesized by microsomes from each line were measured by positivereflectron mode MALDI-TOF MS and normalized, in percent, to the WTpositive control. Boiled wildtype membranes (BWT) indicate backgroundion counts.

FIG. 41 shows SDS-PAGE of plant extracts and protein A or hydroxyapatitepurified samples from MDX-060 LEX^(Opt) under non-reducing (FIG. 41A)and reducing (FIG. 41B) conditions, respectively. MAb purified from aCHO cell line (MDX-060 CHO) was used as a positive control. Mark12molecular weight markers were included on the gels. Gels were stainedwith Colloidal Blue.

FIG. 42 shows the spectra obtained from negative, reflectron modeMALDI-TOF mass spectrometric analysis of 2-AA labeled N-glycans releasedfrom MDX-060 mAbs expressed in CHO (MDX-060 CHO), wild-type Lemna(MDX-060 LEX), or Lemna transformed with the XylT/FucT RNAi construct(MDX-060 LEX^(Opt)). Significant peaks are identified by thecorresponding mass ([M−H]⁻). The * indicates the location of matrixartifacts.

FIG. 43 shows the spectra obtained from NP-HPLC-QTOF MS analysis of 2-AAlabeled N-glycans released from MDX-060 mAbs expressed in CHO (MDX-060CHO), wild-type Lemna (MDX-060 LEX), or Lemna transformed with theXylT/FucT RNAi construct (MDX-060 LEX^(Opt)). 2-AA labeled N-glycanswere separated by normal phase chromatography and detected byfluorescence. The most abundant peaks from each sample (labeled a-i)were characterized by on-line negative mode QTOF MS and theircorresponding QTOF mass spectra ([M-2H]²⁻) are shown.

FIG. 44 shows in vitro activity of MDX-060 mAbs as measured by flowcytometric analysis of MDX-060 CHO, LEX, or glyco-optimized LEX^(Opt)mAb binding to CD30 expressed on L540 cells. L540 cells were incubatedwith increasing concentrations of the indicated antibody as outlined inExample 6 herein below. Geo Mean Fluorescence Intensity (GMFI) isplotted against the various concentrations of mAb used. ▪: MDX-060 CHO;▴: MDX-060 LEX; ▾: MDX-060 LEX^(opt).

FIG. 45 shows equilibrium binding of glyco-optimized and wild-type mAbto two different human FcRγIIIa allotypes (Val¹⁵⁸ or Phe¹⁵⁸). Thebinding signal as a function of FcRγIIIa was fit to a one-site bindingmodel. ▪: MDX-060 CHO; ▴: MDX-060 LEX; ▾: MDX-060 LEX^(opt).

FIG. 46 shows ADCC activity of MDX-060 mAb derived from CHO, LEX(wild-type Lemna glycosylation), or LEX^(Opt) (RNAi transgenic Lemna).Human effector cells from a FcγRIIIaPhe¹⁵⁸ homozygote donor and aFcγRIIIaPhe/Val¹⁵⁸ heterozygote donor were incubated with BATDA-labeledL540 cells at an effector:target ratio of 50:1 in the presence ofincreasing concentrations of the indicated antibodies. Specific percentlysis at each mAb concentration is plotted. Human mAb1 not recognizingantigen on L540 cells was used as an isotype control in all experiments.EC₅₀ values (μg/mL), binding constants and maximal percent lysis werecalculated using GraphPad Prism 3.0 software. ▪: MDX-060 CHO; ▴: MDX-060LEX; ▾: MDX-060 LEX^(opt).

FIG. 47 shows intact mass analysis of the MDX-060 LEX mAb compositionsproduced in wild-type L. minor comprising the MDXA01 construct. WhenXylT and FucT expression are not suppressed in L. minor, therecombinantly produced MDX-060 LEX mAb composition comprises at least 7different glycoforms, with the G0XF³ glycoform being the predominatespecies present. Note the absence of a peak representing the G0glycoform.

FIG. 48 shows glycan mass analysis of the heavy chain of the MDX-060 LEXmAb produced in wild-type L. minor comprising the MDXA01 construct. WhenXylT and FucT expression are not suppressed in L. minor, the predominateN-glycan species present is G0XF³, with additional major peaksreflecting the G0X species. Note the minor presence of the G0 glycanspecies.

FIG. 49 shows intact mass analysis of the MDX-060 LEX^(Opt) mAbcompositions produced in transgenic L. minor comprising the MDXA04construct. When XylT and FucT expression are suppressed in L. minor, theintact mAb composition contains only G0 N-glycans. In addition, thecomposition is substantially homogeneous for the G0 glycoform (peak 2),wherein both glycosylation sites are occupied by the G0 N-glycanspecies, with two minor peaks reflecting trace amounts of precursorglycoforms (peak 1, showing mAb having an Fc region wherein the C_(H)2domain of one heavy chain has a G0 glycan species attached to Asn 297,and the C_(H)2 domain of the other heavy chain is unglycosylated; andpeak 3, showing mAb having an Fc region wherein the Asn 297glycosylation site on each of the C_(H)2 domains has a G0 glycan speciesattached, with a third G0 glycan species attached to an additionalglycosylation site within the mAb structure).

FIG. 50 shows glycan mass analysis of the heavy chain of the MDX-060LEX^(Opt) mAb produced in transgenic L. minor comprising the MDXA04construct. When XylT and FucT expression are suppressed in L. minor, theonly readily detectable N-glycan species attached to the Asn 297glycosylation sites of the C_(H)2 domains of the heavy chains is G0.

FIGS. 51A (MALDI analysis) and 51B (HPLC analysis) show that thehomogeneous glycosylation profile exhibited by mAbI produced intransgenic L. minor (line 24) comprising the mAbI04 RNAi construct wasconsistently observed with scaled-up production. This glycosylationprofile was consistent over the 8-month period of continuous maintenanceof the transgenic line via clonal expansion.

FIG. 52 shows that suppression of FucT and XylT expression using thechimeric RNAi mAbI04 construct of FIG. 12 results in endogenousglycoproteins having a homogeneous glycosylation pattern consistent withthat observed for recombinant glycoproteins. For this figure, theβ1,2-linked xylose residue attached to the trimannose core structure isdesignated by the star symbol.

FIG. 53 shows the structure of complex N-glycans described in Example 6below. M=mannose; Gn=N-acetylglucosamine; A=galactose; X=xylose;F=fucose.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention provides compositions and methods for altering theN-glycosylation pattern of homologous and heterologous polypeptidesproduced in a plant, particularly a plant that serves as an expressionsystem for recombinant proteins of interest. The methods comprise theuse of nucleotide constructs comprising one or more sequences that arecapable of inhibiting expression of α1,3-fucosyltransferase (FucT) andβ1,2-xylosyltransferase (XylT) in a plant. Compositions for use inpracticing the methods of the invention, and compositions comprisingglycoproteins having a substantially homologous glycosylation profile,including glycan-optimized monoclonal antibodies with improved effectorfunction, are also provided.

Definitions

“Polypeptide” refers to any monomeric or multimeric protein or peptide.

“Biologically active polypeptide” refers to a polypeptide that has thecapability of performing one or more biological functions or a set ofactivities normally attributed to the polypeptide in a biologicalcontext. Those skilled in the art will appreciate that the term“biologically active” includes polypeptides in which the biologicalactivity is altered as compared with the native protein (e.g.,suppressed or enhanced), as long as the protein has sufficient activityto be of interest for use in industrial or chemical processes or as atherapeutic, vaccine, or diagnostics reagent. Biological activity can bedetermined by any method available in the art. For example, thebiological activity of members of the interferon family of proteins canbe determined by any of a number of methods including their interactionwith interferon-specific antibodies, their ability to increaseresistance to viral infection, or their ability to modulate thetranscription of interferon-regulated gene targets. In like manner,biological activity of monoclonal antibodies can be determined by any ofa number of methods including, but not limited to, assays for measuringbinding specificity and effector function, for example, using assays forantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC) activity.

By “host cell” is intended a cell that comprises a heterologous nucleicacid sequence of the invention. Though the nucleic acid sequences of theinvention, and fragments and variants thereof, can be introduced intoany cell of interest, of particular interest are plant host cells. Insome embodiments, the plant host cells are cells of a plant that servesas a host for expression of recombinant proteins, for example, a plantexpression system for production of recombinant mammalian proteins ofinterest as noted herein below.

By “heterologous polypeptide of interest” is intended a polypeptide thatis not expressed by the host cells in nature. Conversely, a “homologouspolypeptide” is intended a polypeptide that is naturally produced withinthe cells of the host. Heterologous and homologous polypeptides thatundergo post-translational N-glycosylation are referred to herein asheterologous or homologous glycoproteins. In accordance with the methodsof the present invention, the N-glycosylation pattern of bothheterologous and homologous glycoproteins is altered within the cells ofa plant host so that these glycoproteins have an N-glycosylation patternthat is more similar to that observed with mammalian hosts.

Exemplary heterologous polypeptide and heterologous nucleotide sequencesof interest include, but are not limited to, sequences that encodemammalian polypeptides, such as insulin, growth hormone, α-interferon,β-interferon, β-glucocerebrosidase, β-glucoronidase, retinoblastomaprotein, p53 protein, angiostatin, leptin, erythropoietin (EPO),granulocyte macrophage colony stimulating factor, plasminogen, tissueplasminogen activator, blood coagulation factors, for example, FactorVII, Factor VIII, Factor IX, and activated protein C, alpha1-antitrypsin, monoclonal antibodies (mAbs), Fab fragments, single-chainantibodies, cytokines, receptors, hormones, human vaccines, animalvaccines, peptides, and serum albumin.

For purposes of the present invention, the terms “N-glycan,” “N-linkedglycan,” and “glycan” are used interchangeably and refer to an N-linkedoligosaccharide, e.g., one that is or was attached by anN-acetylglucosamine (GlcNAc) residue linked to the amide nitrogen of anasparagine residue in a protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialicacid (e.g., N-acetyl-neuraminic acid (NeuAc)). The processing of thesugar groups occurs cotranslationally in the lumen of the ER andcontinues in the Golgi apparatus for N-linked glycoproteins.

By “oligomannosidic core structure” or “trimannose core structure” of acomplex N-glycan is intended the core structure shown in FIG. 29A,wherein the core comprises three mannose (Man) and twoN-acetylglucosamine (GlcNAc) monosaccharide residues that are attachedto the asparagine residue of the glycoprotein. The asparagine residue isgenerally within the conserved peptide sequence Asn-Xxx-Thr orAsn-Xxx-Ser, where Xxx is any residue except proline, aspartate, orglutamate. Subsequent glycosylation steps yield the final complexN-glycan structure. The trimannose core structure is denoted herein as“Man₃GlcNAc₂.”

The N-glycans attached to glycoproteins differ with respect to thenumber of branches (antennae) comprising peripheral sugars (e.g.,GlcNAc, galactose, fucose, and sialic acid) that are added to thetrimannose core structure. N-glycans are commonly classified accordingto their branched constituents (e.g., complex, high mannose, or hybrid).A “complex” type N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of a “trimannose” core. Where one GlcNAc is attached to each mannosearm, the species of N-linked glycan is denoted herein as“GlcNAc₂Man₃GlcNAc₂” or “GnGn.” Where only one GlcNac is attached, theN-glycan species is denoted herein as “GlcNAc₁Man₃GlcNAc₂”, wherein theGlcNac is attached to either the 1,3 mannose arm (denoted “MGn” herein)or the 1,6 mannose arm (denoted “GnM” herein) (see FIG. 30). ComplexN-glycans may also have galactose (“Gal”) or N-acetylgalactosamine(“GalNAc”) sugar residues that are optionally modified with sialic acidor derivatives (e.g., “NeuAc,” where “Neu” refers to neuraminic acid and“Ac” refers to acetyl). Where a galactose sugar residue is attached toeach GlcNAc on each mannose arm, the species of N-linked glycan isdenoted herein as “Gal₂GlcNAc₂Man₃GlcNAc₂.” Complex N-glycans may alsohave intrachain substitutions comprising “bisecting” GlcNAc and corefucose (“Fuc”). Complex N-glycans may also have multiple antennae on the“trimannose core,” often referred to as “multiple antennary glycans.” A“high mannose” type N-glycan has five or more mannose residues. A“hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3mannose arm of the trimannose core and zero or more mannoses on the 1,6mannose arm of the trimannose core.

The terms “G0 glycan” and “G0 glycan structure” and “G0 glycan species”are used interchangeably and are intended to mean the complex N-linkedglycan having the GlcNAc₂Man₃GlcNAc₂ structure, wherein no terminalsialic acids (NeuAcs) or terminal galactose (Gal) sugar residues arepresent. If a G0 glycan comprises a fucose (“Fuc”) residue attached tothe trimannose core structure, it is referred to herein as a “G0F³glycan” (having the plant-specific α1,3-linked fucose residue) or “G0F⁶glycan” (having the mammalian α1,6-linked fucose residue). In plants, aG0 glycan comprising the plant-specific β1,2-linked xylose residueattached to the trimannose core structure is referred to herein as a“G0X glycan,” and a G0 glycan comprising both the plant-specificβ1,2-linked xylose residue and plant-specific α1,3-linked fucose residueattached to the trimannose core structure is referred to herein as a“G0XF³ glycan.”

The terms “G1 glycan” and “G1 glycan structure” and “G1 glycan species”are used interchangeably and are intended to mean the complex N-linkedglycan having the GlcNAc₂Man₃GlcNAc₂ structure, wherein one terminalgalactose (Gal) residue is attached to either the 1,3 mannose or 1,6mannose arm, and no terminal sialic acids are present. The terms “G2glycan” and “G2 glycan structure” and “G2 glycan species” are usedinterchangeably and are intended to mean the complex N-linked glycanhaving the GlcNAc₂Man₃GlcNAc₂ structure, wherein a terminal galactose(Gal) residue is attached to the 1,3 mannose arm and the 1,6 mannosearm, and no terminal sialic acids are present.

The term “glycoform” as used herein refers to a glycoprotein containinga particular carbohydrate structure or structures. Thus, for example, a“G0 glycoform” refers to a glycoprotein that comprises only G0 glycanspecies attached to its glycosylation sites. It is recognized that aglycoprotein having more than one glycosylation site can have the sameglycan species attached to each glycosylation site, or can havedifferent glycan species attached to different glycosylation sites. Inthis manner, different patterns of glycan attachment yield differentglycoforms of a glycoprotein.

The term “glycosylation profile” is intended to mean the characteristic“fingerprint” of the representative N-glycan species that have beenreleased from a glycoprotein composition or glycoprotein product, eitherenzymatically or chemically, and then analyzed for their carbohydratestructure, for example, using LC-HPLC, or MALDI-TOF MS, and the like.See, for example, the review in Current Analytical Chemistry, Vol. 1,No. 1 (2005), pp. 28-57; herein incorporated by reference in itsentirety.

The terms “substantially homogeneous,” “substantially uniform,” and“substantial homogeneity” in the context of a glycosylation profile fora glycoprotein composition or glycoprotein product are usedinterchangeably and are intended to mean a glycosylation profile whereinat least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99% of the total N-glycan species within theprofile are represented by one desired N-glycan species, with a traceamount of precursor N-glycan species appearing in the profile. By “traceamount” is intended that any given precursor N-glycan species that ispresent in the glycosylation profile is present at less than 5%,preferably less than 4%, less than 3%, less than 2%, less than 1%, andeven less than 0.5% or even less than 0.1% of the total amount ofN-glycan species appearing in the profile. By “precursor” N-glycanspecies is intended an N-glycan species that is incompletely processed.Examples of precursor N-glycan species present in trace amounts in theglycoprotein compositions or glycoprotein products of the invention, andthus appearing in the glycosylation profiles thereof, are theMan3GlcNAc2, MGn (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the1,3 mannose arm), and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 isattached to the 1,6 mannose arm) precursor N-glycan species describedabove.

Thus, for example, where the desired N-glycan species within aglycoprotein product or composition is G0, a substantially homogeneousglycosylation profile for that product or composition would be onewherein at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or at least 99% of the total amount of N-glycanspecies appearing in the glycosylation profile for the product orcomposition is represented by the G0 glycan species, with a trace amountof precursor N-glycan species appearing in the glycosylation profile.For such a composition, a representative precursor N-glycan speciesappearing in its glycosylation profile would be the Man3GlcNAc2, MGn(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,3 mannose arm),and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,6mannose arm) precursor N-glycan species described above.

The terms “substantially homogeneous,” “substantially uniform,” and“substantial homogeneity” in the context of a glycoprotein compositionor glycoprotein product are used interchangeably and are intended tomean the glycoprotein product or glycoprotein composition wherein atleast 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99% of the glycoprotein present in the product orcomposition is represented by one desired glycoform, with a trace amountof precursor or undesired glycoforms being present in the composition.By “trace amount” is intended that any given precursor or undesiredglycoform that is present in the glycoprotein product or composition ispresent at less than 5%, preferably less than 4%, less than 3%, lessthan 2%, less than 1%, and even less than 0.5% or even less than 0.1% ofthe total glycoprotein. By “precursor” glycoform is intended a glycoformwherein at least one glycosylation site is either unglycosylated, or isoccupied by an N-glycan species that represents a precursor of thedesired N-glycan species, or a glycoform wherein one or more additionalglycosylation sites is present, relative to the desired glycoform, andis occupied by (i.e., has attached thereto) the desired N-glycan speciesor an undesired N-glycan species.

Thus, for example, a substantially homogeneous glycoprotein compositionor product comprising the G0 glycoform is a composition or productwherein at least 80%, 80%, at least 85%, at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least 99% of the glycoprotein present inthe product or composition is represented by the G0 glycoform, whereinall anticipated glycosylation sites are occupied by the G0 glycanspecies, with a trace amount of precursor or undesired glycoforms beingpresent in the composition. In such a composition, a representativeprecursor glycoform would be one in which glycosylation sites areunoccupied, and an exemplary undesired glycoform would be a glycoformhaving a mixture of G0 glycan and G0X or G0XF3 glycan species attachedto its glycosylation sites.

The term “antibody” is used in the broadest sense and covers fullyassembled antibodies, antibody fragments that can bind antigen (e.g.,Fab′, F′(ab)₂, Fv, single chain antibodies, diabodies), and recombinantpeptides comprising the foregoing. Antibodies represent one of the manyglycoproteins contemplated by the methods and compositions of thepresent invention. Derivatives of antibodies are also contemplated bythe present invention. Derivatives include fusion proteins comprising animmunoglobulin or portion thereof, such as an Fc region having a C_(H)2domain.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR) regions. The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.(1991) NIH Publ. No. 91-3242, Vol. I, pages 647-669).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effecter functions, such as Fc receptor(FcR) binding, participation of the antibody in antibody-dependentcellular cytotoxicity (ADCC), opsonization, initiation ofcomplement-dependent cytotoxicity (CDC activity), and mast celldegranulation.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (Zapata et al. (1995) ProteinEng. 8(10):1057-1062); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. In a two-chain Fv species, this regionconsists of a dimer of one heavy- and one light-chain variable domain intight, non-covalent association. In a single-chain Fv species, oneheavy- and one light-chain variable domain can be covalently linked byflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy-chain C_(H)1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments that have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of human immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions. For example, humanIgG1 and IgG3 isotypes mediate antibody-dependent cell-mediatedcytotoxicity (ADCC) activity.

Immunoglobulins have conserved N-linked glycosylation of the Fc regionof each of the two heavy chains. Thus, for example, immunoglobulins ofthe IgG type have glycosylated C_(H)2 domains bearing N-linkedoligosaccharides at asparagine 297 (Asn-297). Different glycoforms ofimmunoglobulins exist depending upon the particular N-glycan speciesattached to each of these two glycosylation sites, and depending uponthe degree to which both sites are glycosylated within an immunoglobulincomposition.

“Nucleotide sequence of interest” as used herein with reference toexpression of heterologous polypeptides refers to any polynucleotidesequence encoding a heterologous polypeptide intended for expression ina host, particularly a plant host, for example, in a higher plant,including members of the dicotyledonaceae and monocotyledonaceae. Forexample, polynucleotide sequences encoding therapeutic (e.g., forveterinary or medical uses) or immunogenic (e.g., for vaccination)polypeptides can be expressed using transformed plant hosts, forexample, duckweed, according to the present invention.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The terms “inhibit,” “inhibition,” and “inhibiting” as used herein referto any decrease in the expression or function of a target gene product,including any relative decrement in expression or function up to andincluding complete abrogation of expression or function of the targetgene product. The term “expression” as used herein in the context of agene product refers to the biosynthesis of that gene product, includingthe transcription and/or translation and/or assembly of the geneproduct. Inhibition of expression or function of a target gene product(i.e., a gene product of interest) can be in the context of a comparisonbetween any two plants, for example, expression or function of a targetgene product in a genetically altered plant versus the expression orfunction of that target gene product in a corresponding wild-type plant.Alternatively, inhibition of expression or function of the target geneproduct can be in the context of a comparison between plant cells,organelles, organs, tissues, or plant parts within the same plant orbetween plants, and includes comparisons between developmental ortemporal stages within the same plant or between plants. Any method orcomposition that down-regulates expression of a target gene product,either at the level of transcription or translation, or down-regulatesfunctional activity of the target gene product can be used to achieveinhibition of expression or function of the target gene product.

The term “inhibitory sequence” encompasses any polynucleotide orpolypeptide sequence that is capable of inhibiting the expression of atarget gene product, for example, at the level of transcription ortranslation, or which is capable of inhibiting the function of a targetgene product. Examples of inhibitory sequences include, but are notlimited to, full-length polynucleotide or polypeptide sequences,truncated polynucleotide or polypeptide sequences, fragments ofpolynucleotide or polypeptide sequences, variants of polynucleotide orpolypeptide sequences, sense-oriented nucleotide sequences,antisense-oriented nucleotide sequences, the complement of a sense- orantisense-oriented nucleotide sequence, inverted regions of nucleotidesequences, hairpins of nucleotide sequences, double-stranded nucleotidesequences, single-stranded nucleotide sequences, combinations thereof,and the like. The term “polynucleotide sequence” includes sequences ofRNA, DNA, chemically modified nucleic acids, nucleic acid analogs,combinations thereof, and the like.

It is recognized that inhibitory polynucleotides include nucleotidesequences that directly (i.e., do not require transcription) orindirectly (i.e., require transcription or transcription andtranslation) inhibit expression of a target gene product. For example,an inhibitory polynucleotide can comprise a nucleotide sequence that isa chemically synthesized or in vitro-produced small interfering RNA(siRNA) or micro RNA (miRNA) that, when introduced into a plant cell,tissue, or organ, would directly, though transiently, silence expressionof the target gene product of interest. Alternatively, an inhibitorypolynucleotide can comprise a nucleotide sequence that encodes aninhibitory nucleotide molecule that is designed to silence expression ofthe gene product of interest, such as sense-orientation RNA, antisenseRNA, double-stranded RNA (dsRNA), hairpin RNA (hpRNA), intron-containinghpRNA, catalytic RNA, miRNA, and the like. In yet other embodiments, theinhibitory polynucleotide can comprise a nucleotide sequence thatencodes a mRNA, the translation of which yields a polypeptide thatinhibits expression or function of the target gene product of interest.In this manner, where the inhibitory polynucleotide comprises anucleotide sequence that encodes an inhibitory nucleotide molecule or amRNA for a polypeptide, the encoding sequence is operably linked to apromoter that drives expression in a plant cell so that the encodedinhibitory nucleotide molecule or mRNA can be expressed.

Inhibitory sequences are designated herein by the name of the targetgene product. Thus, for example, an “α1,3-fucosyltransferase (FucT)inhibitory sequence” would refer to an inhibitory sequence that iscapable of inhibiting the expression of a FucT, for example, at thelevel of transcription and/or translation, or which is capable ofinhibiting the function of a FucT. Similarly, a “β1,2-xylosyltransferase(XylT) inhibitory sequence” would refer to an inhibitory sequence thatis capable of inhibiting the expression of a XylT, at the level oftranscription and/or translation, or which is capable of inhibiting thefunction of a XylT. When the phrase “capable of inhibiting” is used inthe context of a polynucleotide inhibitory sequence, it is intended tomean that the inhibitory sequence itself exerts the inhibitory effect;or, where the inhibitory sequence encodes an inhibitory nucleotidemolecule (for example, hairpin RNA, miRNA, or double-stranded RNApolynucleotides), or encodes an inhibitory polypeptide (i.e., apolypeptide that inhibits expression or function of the target geneproduct), following its transcription (for example, in the case of aninhibitory sequence encoding a hairpin RNA, miRNA, or double-strandedRNA polynucleotide) or its transcription and translation (in the case ofan inhibitory sequence encoding an inhibitory polypeptide), thetranscribed or translated product, respectively, exerts the inhibitoryeffect on the target gene product (i.e., inhibits expression or functionof the target gene product).

The term “introducing” in the context of a polynucleotide, for example,a nucleotide construct of interest, is intended to mean presenting tothe plant the polynucleotide in such a manner that the polynucleotidegains access to the interior of a cell of the plant. Where more than onepolynucleotide is to be introduced, these polynucleotides can beassembled as part of a single nucleotide construct, or as separatenucleotide constructs, and can be located on the same or differenttransformation vectors. Accordingly, these polynucleotides can beintroduced into the host cell of interest in a single transformationevent, in separate transformation events, or, for example, in plants, aspart of a breeding protocol. The methods of the invention do not dependon a particular method for introducing one or more polynucleotides intoa plant, only that the polynucleotide(s) gains access to the interior ofat least one cell of the plant. Methods for introducing polynucleotidesinto plants are known in the art including, but not limited to,transient transformation methods, stable transformation methods, andvirus-mediated methods.

“Transient transformation” in the context of a polynucleotide isintended to mean that a polynucleotide is introduced into the plant anddoes not integrate into the genome of the plant.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a plant is intended the introducedpolynucleotide is stably incorporated into the plant genome, and thusthe plant is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” is intended to mean thata polynucleotide, for example, a nucleotide construct described herein,introduced into a plant integrates into the genome of the plant and iscapable of being inherited by the progeny thereof, more particularly, bythe progeny of multiple successive generations. In some embodiments,successive generations include progeny produced vegetatively (i.e.,asexual reproduction), for example, with clonal propagation. In otherembodiments, successive generations include progeny produced via sexualreproduction. A higher plant host that is “stably transformed” with atleast one nucleotide construct that is capable of inhibiting expressionof a FucT and/or XylT as described herein refers to a higher plant hostthat has the nucleotide construct(s) integrated into its genome, and iscapable producing progeny, either via asexual or sexual reproduction,that also comprise the inhibitory nucleotide construct(s) stablyintegrated into their genome, and hence the progeny will also exhibitthe desired phenotype of having an altered N-glycosylation patterncharacterized by a reduction in the attachment of α1,3-fucose and/orβ1,2-xylose residues to the N-glycans of homologous and heterologousglycoproteins produced therein.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the invention to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, ovules, stems,fruits, leaves, roots, root tips, and the like originating in transgenicplants or their progeny previously transformed with a DNA molecule ofthe invention and therefore consisting at least in part of transgeniccells. As used herein, the term “plant cell” includes, withoutlimitation, cells of seeds, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores.

The class of plants that can be used in the methods of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous (monocot) anddicotyledonous (dicot) plants. Examples of dicots include, but are notlimited to, legumes including soybeans and alfalfa, tobacco, potatoes,tomatoes, and the like. Examples of monocots include, but are notlimited to, maize, rice, oats, barley, wheat, members of the duckweedfamily, grasses, and the like. “Lower-order plants” refers tonon-flowering plants including ferns, horsetails, club mosses, mosses,liverworts, hornworts, algae, for example, red, brown, and green algae,gametophytes, sporophytes of pteridophytes, and the like. In someembodiments, the plant of interest is a member of the duckweed family ofplants.

The term “duckweed” refers to members of the family Lemnaceae. Thisfamily currently is divided into five genera and 38 species of duckweedas follows: genus Lemna (L. aequinoctialis, L. disperma, L.ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L.obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S.punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa.borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa,Wa. microscopica, Wa. neglecta); genus Wolfiella (Wl. caudata, Wl.denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl.rotunda, and Wl. neotropica) and genus Landoltia (L. punctata). Anyother genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention. Lemna species can be classified using thetaxonomic scheme described by Landolt (1986) Biosystematic Investigationon the Family of Duckweeds: The family of Lemnaceae—A Monograph Study(Geobatanischen Institut ETH, Stiftung Rubel, Zurich).

The term “duckweed nodule” as used herein refers to duckweed tissuecomprising duckweed cells where at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the cells are differentiated cells.A “differentiated cell,” as used herein, is a cell with at least onephenotypic characteristic (e.g., a distinctive cell morphology or theexpression of a marker nucleic acid or protein) that distinguishes itfrom undifferentiated cells or from cells found in other tissue types.The differentiated cells of the duckweed nodule culture described hereinform a tiled smooth surface of interconnected cells fused at theiradjacent cell walls, with nodules that have begun to organize into frondprimordium scattered throughout the tissue. The surface of the tissue ofthe nodule culture has epidermal cells connected to each other viaplasmadesmata. Members of the duckweed family reproduce by clonalpropagation, and thus are representative of plants that clonallypropagate.

“Duckweed-preferred codons” as used herein refers to codons that have afrequency of codon usage in duckweed of greater than 17%.

“Lemna-preferred codons” as used herein refers to codons that have afrequency of codon usage in the genus Lemna of greater than 17%.

“Lemna gibba-preferred codons” as used herein refers to codons that havea frequency of codon usage in Lemna gibba of greater than 17% where thefrequency of codon usage in Lemna gibba was obtained from the CodonUsage Database (GenBank Release 113,0; athttp://www.kazusa.or.jp/codon/cgibin/showcodon.cgi?species=Lemna+gibba+[gbpln]).

“Translation initiation codon” refers to the codon that initiates thetranslation of the mRNA transcribed from the nucleotide sequence ofinterest.

“Translation initiation context nucleotide sequence” as used hereinrefers to the identity of the three nucleotides directly 5′ of thetranslation initiation codon.

“Secretion” as used herein refers to translocation of a polypeptideacross both the plasma membrane and the cell wall of a host plant cell.

“Operably linked” as used herein in reference to nucleotide sequencesrefers to multiple nucleotide sequences that are placed in a functionalrelationship with each other. Generally, operably linked DNA sequencesare contiguous and, where necessary to join two protein coding regions,in reading frame.

Isolated Polynucleotides and Polypeptides

The present invention provides isolated polynucleotides and polypeptidesthat are involved in further modification of plant N-linked glycans(also referred to as “N-glycans”), particularly anα1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT)identified in Lemna minor, a member of the duckweed family, and variantsand fragments of these polynucleotides and polypeptides. Inhibition ofthe expression of one or both of these proteins, or biologically activevariants thereof, in a plant that expresses these proteins beneficiallyyields an N-glycosylation pattern that has a reduction in the attachmentof α1,3-fucose and β1,2-xylose residues to glycoprotein N-glycans. Insome embodiments of the invention, the methods disclosed herein providefor complete inhibition of expression of FucT and XylT, yielding anN-glycosylation pattern of glycoproteins produced within a plant whereinthe N-linked glycans are devoid of α1,3-fucose and β1,2-xylose residues.

The full-length cDNA sequence, including 5′- and 3′-UTR, for L. minoralpha 1-3 fucosyltransferase (FucT) is set forth in FIG. 1; see also SEQID NO:1 (open reading frame set forth in SEQ ID NO:2). The predictedamino acid sequence encoded thereby is set forth in SEQ ID NO:3. Atleast two isoforms of the L. minor FucT gene have been identified; thehomology between the isoforms is about 90%. The encoded protein sharessome similarity with other FucTs from other higher plants. See FIG. 2.For example, the L. minor FucT sequence shares approximately 50.1%sequence identity with the Arabidopsis thaliana FucT shown in FIG. 2.

The full-length cDNA sequence, including 5′- and 3′-UTR, for L. minorβ1-2 xylosyltransferase (XylT) (isoform #1) is set forth in FIG. 3; seealso SEQ ID NO:4 (ORF set forth in SEQ ID NO:5). The predicted aminoacid sequence encoded thereby is set forth in SEQ ID NO:6. At least twoisoforms of the L. minor XylT gene have been identified; the homologybetween the isoforms is about 90%. The encoded protein shares somesimilarity with other XylTs from other higher plants. See FIG. 4. Forexample, the L. minor XylT shares approximately 56.4% sequence identitywith the Arabidopsis thaliana XylT shown in FIG. 4. A partial-lengthcDNA sequence, including 3′-UTR, for L. minor β1-2 xylosyltransferase(XylT) (isoform #2) is set forth in FIG. 31; see also SEQ ID NO:19 (ORFset forth in SEQ ID NO:20). The predicted amino acid sequence encodedthereby is set forth in SEQ ID NO:21. The partial-length XylT isoform #2shares high sequence identity with the corresponding region of thefull-length XylT isoform #1, as can be seen from the alignment shown inFIG. 32.

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

The coding sequence for the L. minor FucT gene is set forth asnucleotides (nt) 243-1715 of SEQ ID NO:1 and as SEQ ID NO:2, and theamino acid sequence for the encoded FucT polypeptide is set forth in SEQID NO:3. The coding sequence for the L. minor XylT isoform #1 gene isset forth as nucleotides 63-1592 of SEQ ID NO:4 and as SEQ ID NO:5, andthe amino acid sequence for the encoded XylT polypeptide is set forth inSEQ ID NO:6. The coding sequence for the partial-length L. minor XylTisoform #2 gene is set forth as nucleotides 1-1276 of SEQ ID NO:19 andas SEQ ID NO:20, and the amino acid sequence for the encodedpartial-length XylT polypeptide is set forth in SEQ ID NO:21.

In particular, the present invention provides for isolatedpolynucleotides comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NOS:3, 6, and 21. Further provided arepolypeptides having an amino acid sequence encoded by a polynucleotidedescribed herein, for example those set forth in SEQ ID NOS:1, 2, 4, 5,19, and 20, and fragments and variants thereof. Nucleic acid moleculescomprising the complements of these nucleotide sequences are alsoprovided. It is recognized that the coding sequence for the FucT and/orXylT gene can be expressed in a plant for overexpression of the encodedFucT and/or XylT. However, for purposes of suppressing or inhibiting theexpression of these proteins, the respective nucleotide sequences of SEQID NO:1, 2, 4, 5, 19, and 20 will be used to design constructs forsuppression of expression of the respective FucT and/or XylT protein.Thus, polynucleotides, in the context of suppressing the FucT proteinrefers to the FucT coding sequences and to polynucleotides that whenexpressed suppress or inhibit expression of the FucT gene, for example,via direct or indirect suppression as noted herein below. Similarly,polynucleotides, in the context of suppressing or inhibiting the XylTprotein refers to the XylT coding sequences and to polynucleotides thatwhen expressed suppress or inhibit expression of the XylT gene, forexample, via direct or indirect suppression as noted herein below.

Fragments and variants of the disclosed polynucleotides and proteinsencoded thereby are also encompassed by the present invention. By“fragment” is intended a portion of the FucT or XylT polynucleotide or aportion of the FucT or XylT amino acid sequence encoded thereby.Fragments of a polynucleotide may encode protein fragments that retainthe biological activity of the native protein and hence have FucTactivity or XylT activity as noted elsewhere herein. Alternatively,fragments of a polynucleotide that are useful as hybridization probesgenerally do not encode fragment proteins retaining biological activity.Fragments of a FucT or XylT polynucleotide can also be used to designinhibitory sequences for suppression of expression of the FucT and/orXylT polypeptide. Thus, for example, fragments of a nucleotide sequencemay range from at least about 15 nucleotides, 20 nucleotides, about 50nucleotides, about 100 nucleotides, about 150 nucleotides, about 200nucleotides, about 250 nucleotides, about 300 nucleotides, about 350nucleotides, about 400 nucleotides, about 450 nucleotides, about 500nucleotides, about 550 nucleotides, about 600 nucleotides, about 650nucleotides, about 700 nucleotides, about 750 nucleotides, about 800nucleotides, and up to the full-length polynucleotide encoding theproteins of the invention.

A fragment of a FucT polynucleotide that encodes a biologically activeportion of a FucT protein of the invention will encode at least 15, 25,30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 475, 500 contiguousamino acids, or up to the total number of amino acids present in afull-length FucT protein of the invention (for example, 509 amino acidsfor SEQ ID NO:3). A fragment of a XylT polynucleotide that encodes abiologically active portion of a full-length XylT protein of theinvention will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300,350, 400, 450, 475 contiguous amino acids, or up to the total number ofamino acids present in a full-length XylT protein of the invention (forexample, 490 amino acids for SEQ ID NO:3). A fragment of a XylTpolynucleotide that encodes a biologically active portion of apartial-length XylT protein of the invention will encode at least 15,25, 30, 50, 100, 150, 200, 250, 300, 350, 400 contiguous amino acids, orup to the total number of amino acids present in a partial-length XylTprotein of the invention (for example, 490 amino acids for SEQ ID NO:21)

Thus, a fragment of a FucT or XylT polynucleotide may encode abiologically active portion of a FucT or XylT protein, respectively, orit may be a fragment that can be used as a hybridization probe or PCRprimer, or used to design inhibitory sequences for suppression, usingmethods disclosed below. A biologically active portion of a FucT or XylTprotein can be prepared by isolating a portion of one of the FucT orXylT polynucleotides of the invention, respectively, expressing theencoded portion of the FucT or XylT protein (e.g., by recombinantexpression in vitro), and assessing the activity of the encoded portionof the FucT or XylT polypeptide. Polynucleotides that are fragments ofan FucT or XylT nucleotide sequence comprise at least 15, 20, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800,900, 1,000, 1,100, 1,200, 1,300, 1,400, or 1450 contiguous nucleotides,or up to the number of nucleotides present in a FucT or XylTpolynucleotide disclosed herein (for example, 1865, 1473, 1860, 1530,1282, or 1275 nucleotides for SEQ ID NOS:1, 2, 4, 5, 19, and 20,respectively).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe FucT or XylT polypeptides of the invention. Naturally occurringallelic variants such as these can be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a FucT or XylT proteinof the invention. Generally, variants of a particular polynucleotide ofthe invention (for example, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:19, or SEQ ID NO:20, fragments thereof, andcomplements of these sequences) will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to the FucTor XylT polypeptide of SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:21,respectively, is disclosed. Percent sequence identity between any twopolypeptides can be calculated using sequence alignment programs andparameters described elsewhere herein. Where any given pair ofpolynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore sites in the native protein and/or substitution of one or moreamino acids at one or more sites in the native protein. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, the enzymatic activity of attaching the α1,3-linkedfucose residue (activity of FucT) or β1,2-linked xylose residue(activity of XylT) to glycoprotein N-glycans in plants as describedherein. Such variants may result from, for example, genetic polymorphismor from human manipulation. Biologically active variants of a nativeFucT or XylT protein of the invention will have at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to the amino acid sequencefor the native protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the invention may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the FucT and XylTproteins can be prepared by mutations in the DNA. Methods formutagenesis and polynucleotide alterations are well known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein. Guidance as to appropriate amino acid substitutions thatdo not affect biological activity of the protein of interest may befound in the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal.

Thus, the polynucleotides of the invention include both the naturallyoccurring FucT and XylT sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring FucT andXylT proteins as well as variations and modified forms thereof. Suchvariants will continue to possess the desired activity. Thus, whereexpression of a functional protein is desired, the expressed proteinwill possess the desired FucT or XylT activity, i.e., the enzymaticactivity of attaching the α1,3-linked fucose residue (activity of FucT)or β1,2-linked xylose residue (activity of XylT) to glycoproteinN-glycans in plants as described herein. Where the objective isinhibition of expression or function of the FucT and/or XylTpolypeptide, the desired activity of the variant polynucleotide orpolypeptide is one of inhibiting expression or function of therespective FucT and/or XylT polypeptide. Obviously, where expression ofa functional FucT or XylT variant is desired, the mutations that will bemade in the DNA encoding the variant must not place the sequence out ofreading frame and optimally will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent ApplicationPublication No. 75,444.

Where a functional protein is desired, the deletions, insertions, andsubstitutions of the protein sequences encompassed herein are notexpected to produce radical changes in the characteristics of theprotein. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays, including the assays for monitoring FucT andXylT activity described herein below in the Experimental section.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different FucT or XylTcoding sequences can be manipulated to create a new FucT or XylT proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the FucT or XylTgene of the invention and other known FucT or XylT genes, respectively,to obtain a new gene coding for a protein with an improved property ofinterest. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The comparison of sequences and determination of percent identity andpercent similarity between two sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (1970) J. Mol. Biol. 48:444-453 algorithm, which is incorporatedinto the GAP program in the GCG software package (available atwww.accelrys.com), using either a BLOSSUM62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package, using a BLOSUM62 scoring matrix(see Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA 89:10915) and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. A particularly preferred set of parameters (and the one thatshould be used if the practitioner is uncertain about what parametersshould be applied to determine if a molecule is within a sequenceidentity limitation of the invention) is using a BLOSUM62 scoring matrixwith a gap weight of 60 and a length weight of 3).

The percent identity between two amino acid or nucleotide sequences canalso be determined using the algorithm of Meyers and Miller (1989)CABIOS 4:11-17 which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found, for example, in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and Tijssen (1993)Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic AcidPreparation (Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Ltd., NY, N.Y.).

For purposes of the present invention, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

The FucT and XylT polynucleotides of the invention can be used as probesfor the isolation of corresponding homologous sequences in otherorganisms, more particularly in other plant species. In this manner,methods such as PCR, hybridization, and the like can be used to identifysuch sequences based on their sequence homology to the sequences of theinvention. See, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.) and Innis et al. (1990), PCR Protocols: A Guide toMethods and Applications (Academic Press, New York). Polynucleotidesequences isolated based on their sequence identity to the entire FucTor XylT polynucleotides of the invention (i.e., SEQ ID NOS:1 and 2 forFucT; SEQ ID NOS:4 and 5 for XylT isoform #1 of SEQ ID NO:6; and SEQ IDNOS:19 and 20 for XylT isoform #2 of SEQ ID NO:21) or to fragments andvariants thereof are encompassed by the present invention.

In a PCR method, oligonucleotides primers can be designed for use in PCRreactions for amplification of corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like. Methods for designing PCR primers and PCR cloningare generally known in the art and are disclosed in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York).

In a hybridization method, all or part of a known nucleotide sequencecan be used as a probe that selectively hybridizes to othercorresponding polynucleotides present in a population of cloned genomicDNA fragments or cDNA fragments (i.e., cDNA or genomic libraries) fromanother organism of interest. The so-called hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Probes for hybridization can bemade by labeling synthetic oligonucleotides based on the nucleotidesequence of interest, for example, the FucT or XylT polynucleotides ofthe invention. Degenerate primers designed on the basis of conservednucleotides or amino acid residues in the known nucleotide or encodedamino acid sequence can additionally be used. Methods for constructionof cDNA and genomic libraries, and for preparing hybridization probes,are generally known in the art and are disclosed in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.), herein incorporated byreference.

For example, all or part of the specific known FucT or XylTpolynucleotide sequence may be used as a probe that selectivelyhybridizes to other FucT or XylT nucleotide and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and more optimally at least about 20nucleotides in length. This technique may be used to isolate othercorresponding FucT or XylT nucleotide sequences from a desired organismor as a diagnostic assay to determine the presence of a FucT or XylTcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Innis et al., eds. (1990) PCR Protocols: AGuide to Methods and Applications (Academic Press, New York)).

Thus, in addition to the native FucT and XylT polynucleotides andfragments and variants thereof, the isolated polynucleotides of theinvention also encompass homologous DNA sequences identified andisolated from other organisms by hybridization with entire or partialsequences obtained from the FucT or XylT polynucleotides of theinvention or variants thereof. Conditions that will permit other DNAsequences to hybridize to the DNA sequences disclosed herein can bedetermined in accordance with techniques generally known in the art. Forexample, hybridization of such sequences may be carried out undervarious conditions of moderate, medium, high, or very high stringency asnoted herein above.

Methods of the Invention

The present invention is directed to methods for altering proteinglycosylation patterns in higher plants, particularly in higher plantsthat serve as hosts for production of recombinant proteins, particularlyrecombinant mammalian proteins of pharmaceutical interest. The methodsfind use in producing higher plants that are capable of producingrecombinant proteins having an N-glycosylation pattern that more closelyresembles that found in mammals. Compositions of the invention includehigher plants that are stably transformed to comprise an alteredN-glycosylation pattern of their endogenous (i.e., homologous) andrecombinantly produced heterologous proteins. In some embodiments, thehigher plants are transgenic plants that produce monoclonal antibodies(mAbs) to mammalian proteins that have enhanced ADCC activity relativeto mAbs produced in a control plant that has not had the glycosylationmachinery altered to reduce the plant-specific attachment of α1,3-fucoseresidues to the N-glycans of homologous and heterologous glycoproteinsproduced therein.

The methods of the invention target the suppression (i.e., inhibition)of the expression of one or both of the enzymes involved the productionof complex glycoproteins in higher plants. Of particular interest issuppression of a fucosyltransferase or one or more isoforms thereof,suppression of a xylosyltransferase or one or more isoforms thereof, orsuppression of expression of both of these proteins and one or moreisoforms thereof. It is recognized that suppression of thefucosyltransferase and/or xylosyltransferase and one or more isoformsthereof can be accomplished transiently. Alternatively, by stablysuppressing the expression of the fucosyltransferase and/orxylosyltransferase, it is possible to produce transgenic higher plantsthat carry over from generation to generation, either asexually orsexually, the ability to produce glycoproteins having an N-glycosylationpattern that more closely resembles that found in mammals, moreparticularly, in humans. This advantageously provides for the productionof recombinant mammalian glycoproteins that have reduced attachment ofthe plant β1,2-linked xylose residue and/or α1,3-linked fucose residueto glycoprotein N-glycans.

Inhibition of the expression of one or advantageously both of theseproteins in a plant, for example, a dicotyledonous or monocotyledonousplant, for example, a duckweed plant, can be carried out using anymethod known in the art. In this manner, a polynucleotide comprising aninhibitory sequence for FucT, XylT, or a combination thereof isintroduced into the host cell of interest. For transient suppression,the FucT or XylT inhibitory sequence can be a chemically synthesized orin vitro-produced small interfering RNA (siRNA) or micro RNA (miRNA)that, when introduced into the host cell, would directly, thoughtransiently, inhibit FucT, XylT, or a combination thereof, by silencingexpression of these target gene product(s).

Alternatively, stable suppression of expression of FucT, XylT, or acombination thereof is desirable as noted herein above. Thus, in someembodiments, the activity of the FucT or the XylT polypeptide of theinvention is reduced or eliminated by transforming a plant cell with anexpression cassette that expresses a polynucleotide that inhibits theexpression of the FucT or XylT, or both. The polynucleotide may inhibitthe expression of the FucT or XylT, or both, directly, by preventingtranscription or translation of the FucT or XylT messenger RNA, orindirectly, by encoding a polypeptide that inhibits the transcription ortranslation of a gene encoding the FucT or XylT, or both. Methods forinhibiting or eliminating the expression of a gene in a plant are wellknown in the art, and any such method may be used in the presentinvention to inhibit the expression of FucT or XylT, or both.

Thus, in some embodiments, expression of the FucT and/or XylT proteincan be inhibited by introducing into the plant a nucleotide construct,such as an expression cassette, comprising a sequence that encodes aninhibitory nucleotide molecule that is designed to silence expression ofthe FucT and/or XylT gene product of interest, such as sense-orientationRNA, antisense RNA, double-stranded RNA (dsRNA), hairpin RNA (hpRNA),intron-containing hpRNA, catalytic RNA, miRNA, and the like. In otherembodiments, the nucleotide construct, for example, an expressioncassette, can comprise a sequence that encodes a mRNA, the translationof which yields a polypeptide of interest that inhibits expression orfunction of the FucT and/or XylT gene product of interest. Where thenucleotide construct comprises a sequence that encodes an inhibitorynucleotide molecule or a mRNA for a polypeptide of interest, thesequence is operably linked to a promoter that drives expression in aplant cell so that the encoded inhibitory nucleotide molecule or mRNAcan be expressed.

In accordance with the present invention, the expression of a FucT orXylT gene is inhibited if the protein level of the FucT or XylT isstatistically lower than the protein level of the same FucT or XylT in aplant that has not been genetically modified or mutagenized to inhibitthe expression of that FucT or XylT. In particular embodiments of theinvention, the protein level of the FucT or XylT, or both, in a modifiedplant according to the invention is less than 95%, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, or less than 5% of theprotein level of the same FucT or XylT in a plant that is not a mutantor that has not been genetically modified to inhibit the expression ofthat FucT or XylT, or both the FucT or XylT. The expression level of theFucT or XylT, or both, may be measured directly, for example, byassaying for the level of FucT or XylT, or both, expressed in the plantcell or plant, or indirectly, for example, by observing the effect in atransgenic plant at the phenotypic level, i.e., by transgenic plantanalysis, observed as a reduction, or even elimination, of theattachment of β1,2-xylose and/or α1,3-fucose residues to theglycoprotein N-glycans in the plant.

In other embodiments of the invention, the activity of FucT or XylT, orboth, is reduced or eliminated by transforming a plant cell with anexpression cassette comprising a polynucleotide encoding a polypeptidethat inhibits the activity of FucT or XylT, or both. The activity of aFucT or XylT is inhibited according to the present invention if theactivity of the FucT or XylT is statistically lower than the activity ofthe same FucT or XylT in a plant that has not been genetically modifiedto inhibit the activity of that FucT or XylT. In particular embodimentsof the invention, the activity of the FucT or XylT in a modified plantaccording to the invention is less than 95%, less than 90%, less than80%, less than 70%, less than 60%, less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10%, or less than 5% of the activityof the same FucT or XylT in a plant that has not been geneticallymodified to inhibit the expression of that FucT or XylT. The activity ofa FucT or XylT is “eliminated” according to the invention when it is notdetectable by the assay methods described elsewhere herein.

In other embodiments, the activity of a FucT or XylT, or both, may bereduced or eliminated by disrupting the gene encoding the FucT or XylT,or both of these genes. The invention encompasses mutagenized plants,particularly plants that are members of the duckweed family, that carrymutations in a FucT or XylT gene, or mutations in both genes, where themutations reduce expression of the FucT and/or XylT gene or inhibit theactivity of the encoded FucT and/or XylT.

The methods of the invention can involve any method or mechanism knownin the art for reducing or eliminating the activity or level of FucTand/or XylT in the cells of a higher plant, including, but not limitedto, antisense suppression, sense suppression, RNA interference, directeddeletion or mutation, dominant-negative strategies, and the like. Thus,the methods and compositions disclosed herein are not limited to anymechanism or theory of action and include any method where expression orfunction of FucT and/or XylT is inhibited in the cells of the higherplant of interest, thereby altering the N-glycosylation pattern ofendogenous and heterologous glycoproteins produced in the plant.

For example, in some embodiments, the FucT inhibitory sequence or theXylT inhibitory sequence (or both) is expressed in the senseorientation, wherein the sense-oriented transcripts cause cosuppressionof the expression of one or both of these enzymes. Alternatively, theFucT and/or XylT inhibitory sequence (e.g., full-length sequence,truncated sequence, fragments of the sequence, combinations thereof, andthe like) can be expressed in the antisense orientation and thus inhibitendogenous FucT and/or XylT expression or function by antisensemechanisms.

In yet other embodiments, the FucT and/or XylT inhibitory sequence orsequences are expressed as a hairpin RNA, which comprises both a sensesequence and an antisense sequence. In embodiments comprising a hairpinstructure, the loop structure may comprise any suitable nucleotidesequence including for example 5′ untranslated and/or translated regionsof the gene to be suppressed, such as the 5′ UTR and/or translatedregion of the FucT polynucleotide of SEQ ID NO:1 or 2, or the 5′ UTRand/or translated region of the XylT polynucleotide of SEQ ID NO:4, 5,19, or 20, and the like. In some embodiments, the FucT or XylTinhibitory sequence expressed as a hairpin is encoded by an invertedregion of the FucT or XylT nucleotide sequence. In yet otherembodiments, the FucT and/or XylT inhibitory sequences are expressed asdouble-stranded RNA, where one FucT and/or XylT inhibitory sequence isexpressed in the sense orientation and another complementary sequence isexpressed in the antisense orientation. Double-stranded RNA, hairpinstructures, and combinations thereof comprising FucT nucleotidesequences, XylT nucleotide sequences, or combinations thereof mayoperate by RNA interference, cosuppression, antisense mechanism, anycombination thereof, or by means of any other mechanism that causesinhibition of FucT and/or XylT expression or function.

Thus, many methods may be used to reduce or eliminate the activity of aFucT or XylT, or both of these proteins, and any isoforms thereof. By“isoform” is intended a naturally occurring protein variant of the FucTor XylT protein of interest, where the variant is encoded by a differentgene. Generally, isoforms of a particular FucT or XylT protein ofinterest are encoded by a nucleotide sequence having at least 90%sequence identity to the nucleotide sequence encoding the FucT or XylTprotein of interest. More than one method may be used to reduce oreliminate the activity of a single plant FucT or XylT, and isoformsthereof. Non-limiting examples of methods of reducing or eliminating theactivity of a plant FucT or XylT are given below.

Polynucleotide-Based Methods

In some embodiments of the present invention, a plant cell istransformed with an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of FucT or XylT, or both.The term “expression” as used herein refers to the biosynthesis of agene product, including the transcription and/or translation of the geneproduct. For example, for the purposes of the present invention, anexpression cassette capable of expressing a polynucleotide that inhibitsthe expression of at least one FucT or XylT, or both, is an expressioncassette capable of producing an RNA molecule that inhibits thetranscription and/or translation of at least one FucT or XylT, or both.The “expression” or “production” of a protein or polypeptide from a DNAmolecule refers to the transcription and translation of the codingsequence to produce the protein or polypeptide, while the “expression”or “production” of a protein or polypeptide from an RNA molecule refersto the translation of the RNA coding sequence to produce the protein orpolypeptide.

Examples of polynucleotides that inhibit the expression of a FucT orXylT, or both, are given below.

Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression ofFucT or XylT, or both, may be obtained by sense suppression orcosuppression. For cosuppression, an expression cassette is designed toexpress an RNA molecule corresponding to all or part of a messenger RNAencoding a FucT or XylT, or both, in the “sense” orientation.Overexpression of the RNA molecule can result in reduced expression ofthe native gene. Accordingly, multiple plant lines transformed with thecosuppression expression cassette are screened to identify those thatshow the greatest inhibition of FucT or XylT expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the FucT or XylT, all or part of the 5′ and/or3′ untranslated region of a FucT or XylT transcript, or all or part ofboth the coding sequence and the untranslated regions of a transcriptencoding FucT or XylT. In some embodiments where the polynucleotidecomprises all or part of the coding region for the FucT or XylT protein,the expression cassette is designed to eliminate the start codon of thepolynucleotide so that no protein product will be transcribed.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin et al. (2002) Plant Cell14:1417-1432. Cosuppression may also be used to inhibit the expressionof multiple proteins in the same plant. See, for example, U.S. Pat. No.5,942,657. Methods for using cosuppression to inhibit the expression ofendogenous genes in plants are described in Flavell et al. (1994) Proc.Natl. Acad. Sci. USA 91:3490-3496; Jorgensen et al. (1996) Plant Mol.Biol. 31:957-973; Johansen and Carrington (2001) Plant Physiol.126:930-938; Broin et al. (2002) Plant Cell 14:1417-1432; Stoutjesdijket al. (2002) Plant Physiol. 129:1723-1731; Yu et al. (2003)Phytochemistry 63:753-763; and U.S. Pat. Nos. 5,034,323, 5,283,184, and5,942,657; each of which is herein incorporated by reference. Theefficiency of cosuppression may be increased by including a poly-dTregion in the expression cassette at a position 3′ to the sense sequenceand 5′ of the polyadenylation signal. See, U.S. Patent Publication No.20020048814, herein incorporated by reference. Typically, such anucleotide sequence has substantial sequence identity to the sequence ofthe transcript of the endogenous gene, optimally greater than about 65%sequence identity, more optimally greater than about 85% sequenceidentity, most optimally greater than about 95% sequence identity. See,U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated byreference.

Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofFucT or XylT, or both, may be obtained by antisense suppression. Forantisense suppression, the expression cassette is designed to express anRNA molecule complementary to all or part of a messenger RNA encodingthe FucT or XylT. Overexpression of the antisense RNA molecule canresult in reduced expression of the native gene. Accordingly, multipleplant lines transformed with the antisense suppression expressioncassette are screened to identify those that show the greatestinhibition of FucT or XylT expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the FucT or XylT,all or part of the complement of the 5′ and/or 3′ untranslated region ofthe FucT or XylT transcript, or all or part of the complement of boththe coding sequence and the untranslated regions of a transcriptencoding the FucT or XylT. In addition, the antisense polynucleotide maybe fully complementary (i.e., 100% identical to the complement of thetarget sequence) or partially complementary (i.e., less than 100%identical to the complement of the target sequence) to the targetsequence. Antisense suppression may be used to inhibit the expression ofmultiple proteins in the same plant. See, for example, U.S. Pat. No.5,942,657. Furthermore, portions of the antisense nucleotides may beused to disrupt the expression of the target gene. Generally, sequencesof at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400,450, 500, 550, or greater may be used. Methods for using antisensesuppression to inhibit the expression of endogenous genes in plants aredescribed, for example, in Liu et al. (2002) Plant Physiol.129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of whichis herein incorporated by reference. Efficiency of antisense suppressionmay be increased by including a poly-dT region in the expressioncassette at a position 3′ to the antisense sequence and 5′ of thepolyadenylation signal. See, U.S. Patent Publication No. 20020048814,herein incorporated by reference.

Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of aFucT or XylT, or both, may be obtained by double-stranded RNA (dsRNA)interference. For dsRNA interference, a sense RNA molecule like thatdescribed above for cosuppression and an antisense RNA molecule that isfully or partially complementary to the sense RNA molecule are expressedin the same cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of FucT or XylT expression. Methods for usingdsRNA interference to inhibit the expression of endogenous plant genesare described in Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA95:13959-13964, Liu et al. (2002) Plant Physiol. 129:1732-1743, and WO99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which isherein incorporated by reference.

Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference

In some embodiments of the invention, inhibition of the expression ofFucT or XylT, or both, may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofendogenous genes. See, Waterhouse and Helliwell (2003) Nat. Rev. Genet.4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk et al. (2002) Plant Physiol. 129:1723-1731; and Waterhouseand Helliwell (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNAinterference to inhibit or silence the expression of genes aredescribed, for example, in Chuang and Meyerowitz (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;Pandolfini et al. BMC Biotechnology 3:7, and U.S. Patent Publication No.20030175965; each of which is herein incorporated by reference. Atransient assay for the efficiency of hpRNA constructs to silence geneexpression in vivo has been described by Panstruga et al. (2003) Mol.Biol. Rep. 30:135-140, herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith et al. (2000) Nature 407:319-320.In fact, Smith et al. show 100% suppression of endogenous geneexpression using ihpRNA-mediated interference. Methods for using ihpRNAinterference to inhibit the expression of endogenous plant genes aredescribed, for example, in Smith et al. (2000) Nature 407:319-320;Wesley et al. (2001) Plant J. 27:581-590; Wang and Waterhouse (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse (2003) Methods 30:289-295,and U.S. Patent Publication No. 20030180945, each of which is hereinincorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00904, herein incorporated byreference.

Transcriptional gene silencing (TGS) may be accomplished through use ofhpRNA constructs wherein the inverted repeat of the hairpin sharessequence identity with the promoter region of a gene to be silenced.Processing of the hpRNA into short RNAs which can interact with thehomologous promoter region may trigger degradation or methylation toresult in silencing (Aufsatz et al. (2002) PNAS 99 (Suppl. 4):16499-16506; Mette et al. (2000) EMBO J 19(19):5194-5201).

Expression cassettes that are designed to express an RNA molecule thatforms a hairpin structure are referred to herein as RNAi expressioncassettes. In some embodiments, the RNAi expression cassette is designedin accordance with a strategy outlined in FIG. 28. In such embodiments,an RNAi expression cassette can be designed to suppress the expressionof the individual FucT and XylT genes (i.e., each cassette provides asingle gene knockout), or can be designed to suppress the expression ofboth the FucT and XylT genes (i.e., a single RNAi expression cassetteexpresses an inhibitory molecule that provides for suppression ofexpression of both of these genes). Where the RNAi expression cassettesuppresses expression of both the FucT and XylT genes, it is referred toherein as a “chimeric” RNAi expression cassette. The single-gene andchimeric RNAi expression cassettes can be designed to express largerhpRNA structures or, alternatively, small hpRNA structures, as notedherein below.

Thus, in some embodiments, the RNAi expression cassette is designed toexpress larger hpRNA structures having sufficient homology to the targetmRNA transcript to provide for post-transcriptional gene silencing ofone or both of a FucT and XylT gene. For larger hp RNA structures, thesense strand of the RNAi expression cassette is designed to comprise inthe 5′-to-3′ direction the following operably linked elements: apromoter of interest, a forward fragment of the FucT or XylT genesequence comprising about 500 to about 800 nucleotides (nt) of a sensestrand for FucT or XylT, respectively, a spacer sequence comprisingabout 100 to about 700 nt of any sequence as noted herein below, and areverse fragment of the XylT or FucT gene sequence, wherein the reversefragment comprises the antisense sequence complementary to therespective (i.e., FucT or XylT) forward fragment. Thus, for example, ifa forward fragment is represented by nucleotides “ . . . acttg . . . ”,the corresponding reverse fragment is represented by nucleotides “ . . .caagt . . . ”, and the sense strand for such an RNAi expression cassettewould comprise the following sequence: “5′- . . . acttg . . . nnnn . . .caagt . . . -3′, where “nnnn” represents the spacer sequence.

It is recognized that the forward fragment can comprise a nucleotidesequence that is 100% identical to the corresponding portion of thesense strand for the target FucT or XylT gene sequence, or in thealternative, can comprise a sequence that shares at least 90%, at least95%, or at least 98% sequence identity to the corresponding portion ofthe sense strand for the target FucT or XylT gene to be silenced. Inlike manner, it is recognized that the reverse fragment does not have toshare 100% sequence identity to the complement of the forward fragment;rather it must be of sufficient length and sufficient complementarity tothe forward fragment sequence such that when the inhibitory RNA moleculeis expressed, the transcribed regions corresponding to the forwardfragment and reverse fragment will hybridize to form the base-pairedstem (i.e., double-stranded portion) of the hp RNA structure. By“sufficient length” is intended a length that is at least 10%, at least15%, at least 20%, at least 30%, at least 40% of the length of theforward fragment, more frequently at least 50%, at least 75%, at least90%, or least 95% of the length of the forward fragment. By “sufficientcomplementarity” is intended the sequence of the reverse fragment sharesat least 90%, at least 95%, at least 98% sequence identity with thecomplement of that portion of the forward fragment that will hybridizewith the reverse fragment to form the base-paired stem of the hp RNAstructure. Thus, in some embodiments, the reverse fragment is thecomplement (i.e., antisense version) of the forward fragment.

In designing such an RNAi expression cassette, the lengths of theforward fragment, spacer sequence, and reverse fragments are chosen suchthat the combined length of the polynucleotide that encodes the hpRNAconstruct is about 650 to about 2500 nt, about 750 to about 2500 nt,about 750 to about 2400 nt, about 1000 to about 2400 nt, about 1200 toabout 2300 nt, about 1250 to about 2100 nt, or about 1500 to about 1800.In some embodiments, the combined length of the expressed hairpinconstruct is about 650 nt, about 700 nt, about 750 nt, about 800 nt,about 850 nt, about 900 nt, about 950 nt, about 1000 nt, about 1050 nt,about 1100 nt, about 1150 nt, about 1200 nt, about 1250 nt, about 1300nt, about 1350 nt, about 1400 nt, about 1450 nt, about 1500 nt, about1550 nt, about 1600 nt, about 1650 nt, about 1700 nt, about 1750 nt,about 1800 nt, about 1850 nt, about 1900 nt, about 1950 nt, about 2000nt, about 2050 nt, about 2100 nt, about 2150 nt, about 2200 nt, about2250 nt, about 2300 nt, or any such length between about 650 nt to about2300 nt.

In some embodiments, the forward fragment comprises about 500 to about800 nt, for example, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725,750, 775, or 800 nt of a sense strand for FucT or XylT, for example, ofthe sense strand set forth in SEQ ID NO:1 or 2 (FucT) or SEQ ID NO:4, 5,19, or 20 (XylT); the spacer sequence comprises about 100 to about 700nt, for example, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700nt of any sequence as noted below, and the reverse fragment comprisesthe antisense strand for the forward fragment sequence, or a sequencehaving sufficient length and sufficient complementarity to the forwardfragment sequence.

The spacer sequence can be any sequence that has insufficient homologyto the target gene, i.e., FucT or XylT, and insufficient homology toitself such that the portion of the expressed inhibitory RNA moleculecorresponding to the spacer region fails to self-hybridize, and thusforms the loop of the hairpin RNA structure. In some embodiments, thespacer sequence comprises an intron, and thus the expressed inhibitoryRNA molecule forms an ihpRNA as noted herein above. In otherembodiments, the spacer sequence comprises a portion of the sense strandfor the FucT or XylT gene to be silenced, for example, a portion of thesense strand set forth in SEQ ID NO:1 or 2 (FucT) or SEQ ID NO:4, 5, 19,or 20 (XylT), particularly a portion of the sense strand immediatelydownstream from the forward fragment sequence.

The operably linked promoter can be any promoter of interest thatprovides for expression of the operably linked inhibitory polynucleotidewithin the plant of interest, including one of the promoters disclosedherein below. The regulatory region can comprise additional regulatoryelements that enhance expression of the inhibitory polynucleotide,including, but not limited to, the 5′ leader sequences and 5′ leadersequences plus plant introns discussed herein below.

In one embodiment, the RNAi expression cassette is designed to suppressexpression of the FucT polypeptide of SEQ ID NO:3, a biologically activevariant of the FucT polypeptide of SEQ ID NO:3, or a FucT polypeptideencoded by a sequence having at least 75% sequence identity to thesequence of SEQ ID NO:1 or SEQ ID NO:2, for example, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% sequence identityto the sequence of SEQ ID NO:1 or SEQ ID NO:2. In this manner, the sensestrand of the RNAi expression cassette is designed to comprise in the5′-to-3′ direction the following operably linked elements: a promoter ofinterest; a forward fragment of the FucT gene sequence, wherein theforward fragment comprises nt 255-985 of SEQ ID NO:1; a spacer sequencecomprising about 100 to about 700 nt of any sequence as noted above; anda reverse fragment of the FucT gene sequence, wherein the reversefragment comprises the complement (i.e., antisense version) of nt255-985 of SEQ ID NO:1. In one such embodiment, the spacer sequence isrepresented by nt 986-1444 of SEQ ID NO:1, and the total length of thatportion of the sense strand of the RNAi expression cassettecorresponding to the coding sequence for the hpRNA structure is 1918 nt.Stably transforming a plant with a nucleotide construct comprising thisRNAi expression cassette, for example, the vector shown in FIG. 8,effectively inhibits expression of FucT within the plant cells of theplant in which the hpRNA structure is expressed. In one embodiment, theplant of interest is a member of the duckweed family, for example, amember of the Lemnaceae, and the plant has been stably transformed withthe vector shown in FIG. 8.

In other embodiments of the invention, the RNAi expression cassette isdesigned to suppress expression of the XylT polypeptide of SEQ ID NO:6or SEQ ID NO:21, a biologically active variant of the XylT polypeptideof SEQ ID NO:6 or SEQ ID NO:21, or a XylT polypeptide encoded by asequence having at least 75% sequence identity to the sequence of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20, for example, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% sequenceidentity to the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, orSEQ ID NO:20. In this manner, the sense strand of the RNAi expressioncassette is designed to comprise in the 5′-to-3′ direction the followingoperably linked elements: a promoter of interest; a forward fragment ofthe XylT gene sequence, wherein the forward fragment comprises nt318-1052 of SEQ ID NO:4; a spacer sequence comprising about 100 to about700 nt of any sequence as noted above; and a reverse fragment of theXylT gene sequence, wherein the reverse fragment comprises thecomplement (i.e., antisense version) of nt 318-1052 of SEQ ID NO:4. Inone such embodiment, the spacer sequence is represented by nt 1053-1599of SEQ ID NO:4, and the total length of that portion of the sense strandof the RNAi expression cassette corresponding to the coding sequence forthe hpRNA structure is 2015 nt. Stably transforming a plant with anucleotide construct comprising this RNAi expression cassette, forexample, the vector shown in FIG. 9, effectively inhibits expression ofFucT within the plant cells of the plant in which the hpRNA structure isexpressed. In one embodiment, the plant of interest is a member of theduckweed family, for example, a member of the Lemnaceae, and the planthas been stably transformed with the vector shown in FIG. 9.

In other embodiments, the sense strand of the RNAi expression cassetteis designed to comprise in the 5′-to-3′ direction the following operablylinked elements: a promoter of interest; a forward fragment of the XylTgene sequence, wherein the forward fragment comprises nt 1-734 of SEQ IDNO:19; a spacer sequence comprising about 100 to about 700 nt of anysequence as noted above; and a reverse fragment of the XylT genesequence, wherein the reverse fragment comprises the complement (i.e.,antisense version) of nt 1-734 of SEQ ID NO:19. In one such embodiment,the spacer sequence is represented by nt 735-1282 of SEQ ID NO:19, andthe total length of that portion of the sense strand of the RNAiexpression cassette corresponding to the coding sequence for the hpRNAstructure is 2015 nt. Stably transforming a plant with a nucleotideconstruct comprising this RNAi expression cassette, for example, thevector shown in FIG. 9, effectively inhibits expression of FucT withinthe plant cells of the plant in which the hpRNA structure is expressed.In one embodiment, the plant of interest is a member of the duckweedfamily, for example, a member of the Lemnaceae, and the plant has beenstably transformed with the vector shown in FIG. 9.

In yet other embodiments, larger hpRNA structures can be designed suchthat the antisense and sense sequences are in opposite orientation. Inthis manner, the sense strand of the RNAi expression cassette isdesigned to comprise in the 5′-to-3′ direction the following operablylinked elements: a promoter of interest, the full-length FucT or XylTgene sequence in the antisense orientation, and a forward fragment ofthe FucT or XylT gene sequence comprising the 3′-half of the sequence inthe sense orientation (see FIG. 28, design 1). In this type ofconstruct, the 3′-half of the sequence forms the base-paired (i.e.,double-stranded) stem of the hpRNA, and the 5′-half of the sequence actsas a spacer sequence. Without being bound by any theory or mechanism,the 3′ region of the FucT or XylT sequence is chosen to form thedouble-stranded region of the hpRNA for for this construct because thisregion is relatively conserved among different plant species compared tothe 5′ region.

In one such embodiment, the RNAi expression cassette is designed tosuppress expression of the FucT polypeptide of SEQ ID NO:3, abiologically active variant of the FucT polypeptide of SEQ ID NO:3, or aFucT polypeptide encoded by a sequence having at least 75% sequenceidentity to the sequence of SEQ ID NO:1 or SEQ ID NO:2, for example, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2. In thismanner, the sense strand of the RNAi expression cassette is designed tocomprise in the 5′-to-3′ direction the following operably linkedelements: a promoter of interest; nucleotides 1-1865 of SEQ ID NO:1 inantisense orientation, and nucleotides 950-1865 of SEQ ID NO:1 in thesense orientation. Stably transforming a plant with a nucleotideconstruct comprising this RNAi expression cassette effectively inhibitsexpression of FucT within the plant cells of the plant in which thehpRNA structure is expressed. In one embodiment, the plant of interestis a member of the duckweed family, for example, a member of theLemnaceae.

In another such embodiment, the RNAi expression cassette is designed tosuppress expression of the XylT polypeptide of SEQ ID NO:6 or SEQ IDNO:21, a biologically active variant of the XylT polypeptide of SEQ IDNO:6 or SEQ ID NO:21, or a XylT polypeptide encoded by a sequence havingat least 75% sequence identity to the sequence of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:19, or SEQ ID NO:20, for example, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% sequence identity tothe sequence of SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20. In thismanner, the sense strand of the RNAi expression cassette is designed tocomprise in the 5′-to-3′ direction the following operably linkedelements: a promoter of interest, nucleotides 1-1860 of SEQ ID NO:4 inantisense orientation, and nucleotides 950-1860 of SEQ ID NO:4 in thesense orientation. Stably transforming a plant with a nucleotideconstruct comprising this RNAi expression cassette effectively inhibitsexpression of XylT within the plant cells of the plant in which thehpRNA structure is expressed. In one embodiment, the plant of interestis a member of the duckweed family, for example, a member of theLemnaceae.

In another such embodiment, the sense strand of the RNAi expressioncassette is designed to comprise in the 5′-to-3′ direction the followingoperably linked elements: a promoter of interest, nucleotides 1-1282 ofSEQ ID NO:19 in antisense orientation, and nucleotides 652-1282 of SEQID NO:19 in the sense orientation. Stably transforming a plant with anucleotide construct comprising this RNAi expression cassetteeffectively inhibits expression of XylT within the plant cells of theplant in which the hpRNA structure is expressed. In one embodiment, theplant of interest is a member of the duckweed family, for example, amember of the Lemnaceae.

Where suppression of both the FucT and XylT proteins is desired, it canbe achieved by introducing these single-gene RNAi expression cassettesinto the plant in a single transformation event, for example, byassembling these single-gene RNAi expression cassettes within a singletransformation vector, for example, a vector similar to that shown inFIG. 11, or as separate co-transformation events, for example, byassembling these single-gene RNAi expression cassettes within twotransformation vectors, for example, vectors similar to those shown inFIGS. 8 and 9, using any suitable transformation method known in theart, including but not limited to the transformation methods disclosedelsewhere herein.

Alternatively, suppression of both the FucT and XylT proteins can beachieved by introducing into the higher plant of interest a chimericRNAi expression cassette as noted herein above. Thus, in someembodiments of the invention, the sense strand of a chimeric RNAiexpression cassette is designed to comprise in the 5′-to-3′ directionthe following operably linked elements: a promoter of interest; achimeric forward fragment, comprising about 500 to about 650 nucleotides(nt) of a sense strand for FucT and about 500 to about 650 nt of a sensestrand for XylT, wherein the FucT sequence and XylT sequence can be ineither order; a spacer sequence comprising about 100 to about 700 nt ofany sequence; and a reverse fragment of the chimeric forward fragment,wherein the reverse fragment comprises the antisense sequencecomplementary to the respective chimeric forward fragment.

As previously noted for the individual RNAi expression cassettes, it isrecognized that the individual FucT or XylT sequence within the chimericforward fragment can comprise a nucleotide sequence that is 100%identical to the corresponding portion of the sense strand for thetarget FucT and XylT gene sequence, respectively, or in the alternative,can comprise a sequence that shares at least 90%, at least 95%, or atleast 98% sequence identity to the corresponding portion of the sensestrand for the target FucT or XylT gene to be silenced. In like manner,it is recognized that the reverse fragment does not have to share 100%sequence identity to the complement of the chimeric forward fragment;rather it must be of sufficient length and sufficient complementarity tothe chimeric forward fragment sequence, as defined herein above, suchthat when the inhibitory RNA molecule is expressed, the transcribedregions corresponding to the chimeric forward fragment and reversefragment will hybridize to form the base-paired stem (i.e.,double-stranded portion) of the hpRNA structure.

In designing such a chimeric RNAi expression cassette, the lengths ofthe forward fragment, spacer sequence, and reverse fragments are chosensuch that the combined length of the polynucleotide that encodes thehpRNA structure is about 1200 to about 3300 nt, about 1250 to about 3300nt, about 1300 to about 3300 nt, about 1350 to about 3300 nt, about 1400to about 3300 nt, about 1450 nt to about 3300 nt, about 1500 to about3300 nt, about 2200 to about 3100 nt, about 2250 to about 2800 nt, orabout 2500 to about 2700 nt. In some embodiments, the combined length ofthe expressed hairpin construct is about 1200 nt, about 1250 nt, about1300 nt, about 1350 nt, about 1400 nt, about 1450 nt, about 1500 nt,about 1800 nt, about 2200 nt, about 2250 nt, about 2300 nt, about 2350nt, about 2400 nt, about 2450 nt, about 2500 nt, about 2550 nt, about2600 nt, about 2650 nt, about 2700 nt, about 2750 nt, about 2800 nt,about 2850 nt, about 2900 nt, about 2950 nt, about 3000 nt, about 3050nt, about 3100 nt, about 3150 nt, about 3200 nt, about 3250 nt, about3300 nt, or any such length between about 1200 nt to about 3300 nt.

In some embodiments, the chimeric forward fragment comprises about 500to about 650 nt, for example, 500, 525, 550, 575, 600, 625, or 650 nt,of a sense strand for FucT, for example, of the sense strand set forthin SEQ ID NO:1 or 2, and about 500 to about 650 nt, for example, 500,525, 550, 575, 600, 625, or 650 nt, of a sense strand for XylT, forexample, of the sense strand set forth in SEQ ID NO:4, 5, 19, or 20,where the FucT and XylT sequence can be fused in either order; thespacer sequence comprises about 100 to about 700 nt, for example, 100,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, or 700 nt of any sequence of interest; andthe reverse fragment comprises the antisense strand for the chimericforward fragment sequence, or a sequence having sufficient length andsufficient complementarity to the chimeric forward fragment sequence.

As noted above for the single-gene RNAi expression cassettes, the spacersequence can be any sequence that has insufficient homology to thetarget gene, i.e., FucT or XylT, and insufficient homology to itselfsuch that the portion of the expressed inhibitory RNA moleculecorresponding to the spacer region fails to self-hybridize, and thusforms the loop of the hpRNA structure. In some embodiments, the spacersequence comprises an intron, and thus the expressed inhibitory RNAmolecule forms an ihpRNA as noted herein above. In other embodiments,the spacer sequence comprises a portion of the sense strand for the FucTor XylT gene to be silenced, for example, a portion of the sense strandset forth in SEQ ID NO:1 or 2 (FucT) or SEQ ID NO:4, 5, 19, or 20(XylT). In one embodiment, the chimeric forward fragment comprises theFucT and XylT sequence fused in that order, and the spacer sequencecomprises a portion of the XylT sense strand immediately downstream fromthe XylT sequence contained within the chimeric forward fragment. Inanother embodiment, the chimeric forward fragment comprises the XylT andFucT sequence fused in that order, and the spacer sequence comprises aportion of the FucT sense strand immediately downstream from the FucTsequence contained within the chimeric forward fragment.

In some embodiments, the chimeric RNAi expression cassette is designedto suppress expression of the FucT polypeptide of SEQ ID NO:3, abiologically active variant of the FucT polypeptide of SEQ ID NO:3, or aFucT polypeptide encoded by a sequence having at least 75% sequenceidentity to the sequence of SEQ ID NO:1 or SEQ ID NO:2, for example, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2, and tosuppress expression of the XylT polypeptide of SEQ ID NO:6 or SEQ IDNO:21, a biologically active variant of the XylT polypeptide of SEQ IDNO:6 or SEQ ID NO:21, or a XylT polypeptide encoded by a sequence havingat least 75% sequence identity to the sequence of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:19, or SEQ ID NO:20, for example, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% sequence identity tothe sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20.For some of these embodiments, the FucT sequence within the chimericforward fragment is chosen such that it corresponds to nt 700 to nt 1400of SEQ ID NO:1 or SEQ ID NO:2, and/or the XylT sequence within thechimeric forward fragment is chosen such that it corresponds to nt 700to nt 1400 of SEQ ID NO:4 or SEQ ID NO:5, or nt 383 to 1083 of SEQ IDNO:19 or 20. Without being bound by theory, it is believed that thisregion (particularly for FucT) is relatively conserved among differentplant species, and therefore is a potentially good target.

In other embodiments, the sense strand of the chimeric RNAi expressioncassette is designed to comprise in the 5′-to-3′ direction the followingoperably linked elements: a promoter of interest; a chimeric forwardfragment comprising nt 254-855 of SEQ ID NO:1 (FucT sequence) and nt318-943 of SEQ ID NO:4 (XylT sequence); a spacer sequence comprisingabout 100 to about 700 nt of any sequence as noted above; and a reversefragment comprising the complement (i.e., antisense version) of thechimeric forward fragment, i.e., comprising the complement of nt 318-943of SEQ ID NO:4 and the complement of nt 254-855 of SEQ ID NO:1. In aparticular embodiment, the spacer sequence within this chimeric RNAiexpression cassette is represented by nt 944-1443 of SEQ ID NO:4, andthe total length of that portion of the sense strand of the RNAiexpression cassette corresponding to the coding sequence for the hpRNAstructure is 2956 nt.

In another such embodiment, the sense strand of the chimeric RNAiexpression cassette is designed to comprise in the 5′-to-3′ directionthe following operably linked elements: a promoter of interest; achimeric forward fragment comprising nt 318-943 of SEQ ID NO:4 (XylTsequence) and nt 254-855 of SEQ ID NO:1 (FucT sequence); a spacersequence comprising about 100 to about 700 nt of any sequence as notedabove; and a reverse fragment comprising the complement (i.e., antisenseversion) of the chimeric forward fragment, i.e., comprising thecomplement of nt 254-855 of SEQ ID NO:1 and the complement of nt 318-943of SEQ ID NO:4. In a particular embodiment, the spacer sequence withinthis chimeric RNAi expression cassette is represented by nt 856-1355 ofSEQ ID NO:1, and the total length of that portion of the sense strand ofthe RNAi expression cassette corresponding to the coding sequence forthe hpRNA structure is 2956 nt.

In yet other embodiments, the sense strand of the chimeric RNAiexpression cassette is designed to comprise in the 5′-to-3′ directionthe following operably linked elements: a promoter of interest; achimeric forward fragment comprising nt 254-855 of SEQ ID NO:1 (FucTsequence) and nt 1-626 of SEQ ID NO:19 (XylT sequence); a spacersequence comprising about 100 to about 700 nt of any sequence as notedabove; and a reverse fragment comprising the complement (i.e., antisenseversion) of the chimeric forward fragment, i.e., comprising thecomplement of nt 1-626 of SEQ ID NO:19 and the complement of nt 254-855of SEQ ID NO:1. In a particular embodiment, the spacer sequence withinthis chimeric RNAi expression cassette is represented by nt 627-1126 ofSEQ ID NO:19, and the total length of that portion of the sense strandof the RNAi expression cassette corresponding to the coding sequence forthe hpRNA structure is 2956 nt.

In another such embodiment, the sense strand of the chimeric RNAiexpression cassette is designed to comprise in the 5′-to-3′ directionthe following operably linked elements: a promoter of interest; achimeric forward fragment comprising nt 1-626 of SEQ ID NO:19 (XylTsequence) and nt 254-855 of SEQ ID NO:1 (FucT sequence); a spacersequence comprising about 100 to about 700 nt of any sequence as notedabove; and a reverse fragment comprising the complement (i.e., antisenseversion) of the chimeric forward fragment, i.e., comprising thecomplement of nt 254-855 of SEQ ID NO:1 and the complement of nt 1-626of SEQ ID NO:19. In a particular embodiment, the spacer sequence withinthis chimeric RNAi expression cassette is represented by nt 856-1355 ofSEQ ID NO:1, and the total length of that portion of the sense strand ofthe RNAi expression cassette corresponding to the coding sequence forthe hpRNA structure is 2956 nt.

Stably transforming a plant with a nucleotide construct comprising achimeric RNAi expression cassette described herein, for example, stabletransformation with the vector shown in FIG. 10, effectively inhibitsexpression of both FucT and XylT within the plant cells of the plant inwhich the hpRNA structure is expressed. In one embodiment, the plant ofinterest is a member of the duckweed family, for example, a member ofthe Lemnaceae, and the plant has been stably transformed with the vectorshown in FIG. 10.

It is recognized that the plant can be stably transformed with at leasttwo of these chimeric RNAi expression cassettes to provide for veryefficient gene silencing of the FucT and XylT proteins, includingsilencing of any isoforms of these two proteins. See, for example, thetwo orientations provided in “possible design 2” of FIG. 28. In thismanner, the plant can be stably transformed with a first chimeric RNAiexpression cassette wherein the chimeric forward fragment comprises theFucT and XylT sequence fused in that order, and the spacer sequencecomprises a portion of the XylT sense strand immediately downstream fromthe XylT sequence contained within the chimeric forward fragment; andwith a second chimeric RNAi expression cassette wherein the chimericforward fragment comprises the XylT and FucT sequence fused in thatorder, and the spacer sequence comprises a portion of the FucT sensestrand immediately downstream from the FucT sequence contained withinthe chimeric forward fragment.

The operably linked promoter within any of the RNAi expression cassettesencoding large hpRNA structures, or large ihpRNA structures can be anypromoter of interest that provides for expression of the operably linkedinhibitory polynucleotide within the plant of interest, including one ofthe promoters disclosed herein below. The regulatory region can compriseadditional regulatory elements that enhance expression of the inhibitorypolynucleotide, including, but not limited to, the 5′ leader sequencesand 5′ leader sequences plus plant introns discussed herein below.

In yet other embodiments, the RNAi expression cassette can be designedto provide for expression of small hpRNA structures having a base-pairedstem region comprising about 200 base pairs or less. Expression of thesmall hpRNA structure is preferably driven by a promoter recognized byDNA-dependent RNA polymerase III. See, for example, U.S. PatentApplication No. 20040231016, herein incorporated by reference in itsentirety.

In this manner, the RNAi expression cassette is designed such that thetranscribed DNA region encodes an RNA molecule comprising a sense andantisense nucleotide region, where the sense nucleotide sequencecomprises about 19 contiguous nucleotides having about 90% to about 100%sequence identity to a nucleotide sequence of about 19 contiguousnucleotides from the RNA transcribed from the gene of interest and wherethe antisense nucleotide sequence comprises about 19 contiguousnucleotides having about 90% to about 100% sequence identity to thecomplement of a nucleotide sequence of about 19 contiguous nucleotidesof the sense sequence. The sense and antisense nucleotide sequences ofthe RNA molecule should be capable of forming a base-paired (i.e.,double-stranded) stem region of RNA of about 19 to about 200nucleotides, alternatively about 21 to about 90 or 100 nucleotides, oralternatively about 40 to about 50 nucleotides in length. However, thelength of the base-paired stem region of the RNA molecule may also beabout 30, about 60, about 70 or about 80 nucleotides in length. Wherethe base-paired stem region of the RNA molecule is larger than 19nucleotides, there is only a requirement that there is at least onedouble-stranded region of about 19 nucleotides (wherein there can beabout one mismatch between the sense and antisense region) the sensestrand of which is “identical” (allowing for one mismatch) with 19consecutive nucleotides of the target FucT or XylT polynucleotide ofinterest. The transcribed DNA region of this type of RNAi expressioncassette may comprise a spacer sequence positioned between the sense andantisense encoding nucleotide region. The spacer sequence is not relatedto the targeted FucT or XylT polynucleotide, and can range in lengthfrom 3 to about 100 nucleotides or alternatively from about 6 to about40 nucleotides. This type of RNAi expression cassette also comprises aterminator sequence recognized by the RNA polymerase III, the sequencebeing an oligo dT stretch, positioned downstream from theantisense-encoding nucleotide region of the cassette. By “oligo dTstretch” is a stretch of consecutive T-residues. It should comprise atleast 4 T-residues, but obviously may contain more T-residues.

It is recognized that in designing the short hpRNA, the fragments of thetargeted gene sequence (i.e., fragments of FucT or XylT gene sequence)and any spacer sequence to be included within the hpRNA-encoding portionof the RNAi expression cassette are chosen to avoid GC-rich sequences,particularly those with three consecutive G/C's, and to avoid theoccurrence of four or more consecutive T's or A's, as the string “TTTT .. . ” serves as a terminator sequence recognized by the RNA polymeraseIII.

Thus, where gene silencing with a short hpRNA is desired, the RNAiexpression cassette can be designed to comprise in the 5′-to-3′direction the following operably linked elements: a promoter recognizedby a DNA dependent RNA polymerase III of the plant cell, as definedherein below; a DNA fragment comprising a sense and antisense nucleotidesequence, wherein the sense nucleotide sequence comprises at least 19contiguous nucleotides having about 90% to about 100% sequence identityto a nucleotide sequence of at least 19 contiguous nucleotides from thesense strand of the FucT or XylT gene of interest, and wherein theantisense nucleotide sequence comprises at least 19 contiguousnucleotides having about 90% to about 100% sequence identity to thecomplement of a nucleotide sequence of at least 19 contiguousnucleotides of the sense sequence, wherein the sense and antisensenucleotide sequence are capable of forming a double-stranded RNA ofabout 19 to about 200 nucleotides in length; and an oligo dT stretchcomprising at least 4 consecutive T-residues.

In some embodiments of the invention, the RNAi expression cassette isdesigned to express a small hpRNA that suppresses expression of the FucTpolypeptide of SEQ ID NO:3, a biologically active variant of the FucTpolypeptide of SEQ ID NO:3, or a FucT polypeptide encoded by a sequencehaving at least 90% sequence identity to the sequence of SEQ ID NO:1 orSEQ ID NO:2. In this manner, the RNAi expression cassette can bedesigned to comprise in the 5′-to-3′ direction the following operablylinked elements: a promoter recognized by a DNA dependent RNA polymeraseIII of the plant cell, as defined herein below; a DNA fragmentcomprising a sense and antisense nucleotide sequence, wherein the sensenucleotide sequence comprises at least 19 contiguous nucleotides havingabout 90% to about 100% sequence identity to a nucleotide sequence of atleast 19 contiguous nucleotides of SEQ ID NO:1, and wherein theantisense nucleotide sequence comprises at least 19 contiguousnucleotides having about 90% to about 100% sequence identity to thecomplement of a nucleotide sequence of at least 19 contiguousnucleotides of the sense sequence, wherein the sense and antisensenucleotide sequence are capable of forming a double-stranded RNA ofabout 19 to about 200 nucleotides in length; and an oligo dT stretchcomprising at least 4 consecutive T-residues.

In other embodiments of the invention, the RNAi expression cassette isdesigned to express a small hpRNA that suppresses expression of the XylTpolypeptide of SEQ ID NO:6 or SEQ ID NO:21, a biologically activevariant of the XylT polypeptide of SEQ ID NO:6 or SEQ ID NO:21, or aXylT polypeptide encoded by a sequence having at least 90% sequenceidentity to the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, orSEQ ID NO:20. In this manner, the RNAi expression cassette can bedesigned to comprise in the 5′-to-3′ direction the following operablylinked elements: a promoter recognized by a DNA dependent RNA polymeraseIII of the plant cell, as defined herein below; a DNA fragmentcomprising a sense and antisense nucleotide sequence, wherein the sensenucleotide sequence comprises at least 19 contiguous nucleotides havingabout 90% to about 100% sequence identity to a nucleotide sequence of atleast 19 contiguous nucleotides of SEQ ID NO:4, and wherein theantisense nucleotide sequence comprises at least 19 contiguousnucleotides having about 90% to about 100% sequence identity to thecomplement of a nucleotide sequence of at least 19 contiguousnucleotides of the sense sequence, wherein the sense and antisensenucleotide sequence are capable of forming a double stranded RNA ofabout 19 to about 200 nucleotides in length; and an oligo dT stretchcomprising at least 4 consecutive T-residues.

Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for FucT or XylT, or both). Methods ofusing amplicons to inhibit the expression of endogenous plant genes aredescribed, for example, in Angell and Baulcombe (1997) EMBO J.16:3675-3684, Angell and Baulcombe (1999) Plant J. 20:357-362, and U.S.Pat. No. 6,646,805, each of which is herein incorporated by reference.

Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of FucT or XylT, or both. Thus, thepolynucleotide causes the degradation of the endogenous messenger RNA,resulting in reduced expression of the FucT or XylT, or both. Thismethod is described, for example, in U.S. Pat. No. 4,987,071, hereinincorporated by reference.

Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression ofFucT or XylT, or both, may be obtained by RNA interference by expressionof a gene encoding a micro RNA (miRNA). miRNAs are regulatory agentsconsisting of about 22 ribonucleotides. miRNA are highly efficient atinhibiting the expression of endogenous genes. See, for example Javieret al. (2003) Nature 425: 257-263, herein incorporated by reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of FucT or XylT expression, the22-nucleotide sequence is selected from a FucT or XylT transcriptsequence and contains 22 nucleotides of said FucT or XylT sequence insense orientation and 21 nucleotides of a corresponding antisensesequence that is complementary to the sense sequence. miRNA moleculesare highly efficient at inhibiting the expression of endogenous genes,and the RNA interference they induce is inherited by subsequentgenerations of plants.

Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding a FucT or XylT, or both, resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a FucT or XylT gene. In otherembodiments, the zinc finger protein binds to a messenger RNA encoding aFucT or XylT and prevents its translation. Methods of selecting sitesfor targeting by zinc finger proteins have been described, for example,in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteinsto inhibit the expression of genes in plants are described, for example,in U.S. Patent Publication No. 20030037355; each of which is hereinincorporated by reference.

Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one FucT or XylT, and reduces theactivity of the FucT or XylT. In another embodiment, the binding of theantibody results in increased turnover of the antibody-FucT or XylTcomplex by cellular quality control mechanisms. The expression ofantibodies in plant cells and the inhibition of molecular pathways byexpression and binding of antibodies to proteins in plant cells are wellknown in the art. See, for example, Conrad and Sonnewald (2003) NatureBiotech. 21:35-36, incorporated herein by reference.

Gene Disruption

In some embodiments of the present invention, the activity of FucT orXylT, or both, is reduced or eliminated by disrupting the gene encodingthe FucT or XylT, or both. The gene encoding the FucT or XylT, or both,may be disrupted by any method known in the art. For example, in oneembodiment, the gene is disrupted by transposon tagging. In anotherembodiment, the gene is disrupted by mutagenizing plants using random ortargeted mutagenesis, and selecting for plants that have reduced FucTand/or XylT activity.

Transposon Tagging

In one embodiment of the invention, transposon tagging is used to reduceor eliminate the activity of FucT or XylT, or both. Transposon taggingcomprises inserting a transposon within an endogenous FucT or XylT geneto reduce or eliminate expression of the FucT or XylT. “FucT” or “XylT”gene is intended to mean the gene that encodes a FucT or XylT,respectively, according to the invention.

In this embodiment, the expression of FucT or XylT is reduced oreliminated by inserting a transposon within a regulatory region orcoding region of the gene encoding the FucT or XylT. A transposon thatis within an exon, intron, 5′ or 3′ untranslated sequence, a promoter,or any other regulatory sequence of a FucT or XylT, or both, gene may beused to reduce or eliminate the expression and/or activity of theencoded FucT or XylT.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes et al. (1999) Trends Plant Sci.4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179:53-59;Meissner et al. (2000) Plant J. 22:265-274; Phogat et al. (2000) J.Biosci. 25:57-63; Walbot (2000) Curr. Opin. Plant Biol. 2:103-107; Gaiet al. (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice et al. (1999)Genetics 153:1919-1928). In addition, the TUSC process for selecting Muinsertions in selected genes has been described in Bensen et al. (1995)Plant Cell 7:75-84; Mena et al. (1996) Science 274:1537-1540; each ofwhich is herein incorporated by reference.

The invention encompasses additional methods for reducing or eliminatingthe activity of FucT or XylT. Examples of other methods for altering ormutating a genomic nucleotide sequence in a plant are known in the artand include, but are not limited to, the use of RNA:DNA vectors, RNA:DNAmutational vectors, RNA:DNA repair vectors, mixed-duplexoligonucleotides, self-complementary RNA:DNA oligonucleotides, andrecombinogenic oligonucleobases. Such vectors and methods of use areknown in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181;5,756,325; 5,760,012; 5,795,972; and 5,871,984; each of which are hereinincorporated by reference. See also, WO 98/49350, WO 99/07865, WO99/25821, and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA96:8774-8778; each of which is herein incorporated by reference.

Thus inhibition of expression of FucT and/or XylT in a higher plant ofinterest can be accomplished by any of the foregoing methods in order toalter the N-glycosylation pattern of endogenous and heterologousglycoproteins produced within that plant such that these glycoproteinscomprise complex N-glycans that have a reduction in the amount ofβ1,2-linked xylose residues and/or α1,3-linked fucose residues. Theextent to which attachment of the β1,2-linked xylose residue and/orα1,3-linked fucose residue to glycoprotein N-glycans is reduced isgoverned by the degree of inhibition of expression of the respectiveXylT and FucT enzymes.

In some embodiments of the invention, recombinant glycoproteins producedin a plant host that is stably transformed using the methods describedherein to target XylT expression have N-linked glycans comprising lessthan 50%, less than 40%, less than 30% of the β1,2-linked xyloseresidues occurring in the respective N-linked glycans of glycloproteinsproduced in a plant host that has not been genetically modified toinhibit expression of the XylT enzyme and isoforms thereof. In otherembodiments, these recombinant glycoproteins have N-linked glycanscomprising less than 25%, less than 20%, less than 15%, less than 10%,less than 5%, or less than 1% of the β1,2-linked xylose residuesoccurring in the respective N-linked glycans of glycloproteins producedin a plant host that has not been genetically modified to inhibitexpression of the XylT enzyme and isoforms thereof. In yet otherembodiments, the methods of the invention provide for complete silencingof the XylT gene and any isoforms thereof within the stably transformedplant, such that the recombinant glycoproteins produced within the planthave N-linked glycans that are devoid of β1,2-linked xylose residues.

In like manner, where a plant host has been stably transformed using themethods described herein to target FucT expression, recombinantglycoproteins produced within the plant have N-linked glycans comprisingless than 50%, less than 40%, less than 30% of the α1,3-linked fucoseresidues occurring in the respective N-linked glycans of glycoproteinsproduced in a plant host that has not been genetically modified toinhibit expression of the FucT enzyme and isoforms thereof. In otherembodiments, these recombinant glycoproteins have N-linked glycanscomprising less than 25%, less than 20%, less than 15%, less than 10%,less than 5%, or less than 1% of the α1,3-linked fucose residuesoccurring in the respective N-linked glycans of glycoproteins producedin a plant host that has not been genetically modified to inhibitexpression of the FucT enzyme and isoforms thereof. In yet otherembodiments, the methods of the invention provide for complete silencingof the FucT gene and any isoforms thereof within the stably transformedplant, such that the recombinant glycoproteins produced within the planthave N-linked glycans that are devoid of α1,3-linked fucose residues.

Where a plant host has been stably transformed using the methodsdescribed herein to target expression of both the XylT and FucT enzymes,and any isoforms thereof, recombinant glycoproteins produced within theplant have N-linked glycans comprising less than 50%, less than 40%,less than 30% of the β-1,2-linked xylose residues and less than 50%,less than 40%, less than 30% of the α1,3-linked fucose residuesoccurring in the respective N-linked glycans of glycoproteins producedin a plant host that has not been genetically modified to inhibitexpression of the XylT and FucT enzymes and isoforms thereof. In otherembodiments, these recombinant glycoproteins have N-linked glycanscomprising less than 25%, less than 20%, less than 15%, less than 10%,less than 5%, or less than 1% of the β1,2-linked xylose residues and25%, less than 20%, less than 15%, less than 10%, less than 5%, or lessthan 1% of the α1,3-linked fucose residues occurring in the respectiveN-linked glycans of glycoproteins produced in a plant host that has notbeen genetically modified to inhibit expression of the XylT and FucTenzymes and isoforms thereof. In yet other embodiments, the methods ofthe invention provide for complete silencing of the XylT and FucT geneand any isoforms thereof within the stably transformed plant, such thatthe recombinant glycoproteins produced within the plant have N-linkedglycans that are devoid of β1,2-linked xylose residues and α1,3-linkedfucose residues.

In some embodiments of the present invention, a plant host that has beenstably transformed using the methods described herein to targetexpression of both the XylT and FucT enzymes, and any isoforms thereof,is capable of producing recombinant glycoproteins wherein the N-linkedglycans are substantially homogenous. By “substantially homogenous” isintended that the glycosylation profile reflects the presence of asingle major peak corresponding to a desired N-glycan species, moreparticularly, the G0 glycan species, wherein at least 90% of theN-glycan structures present on said glycoproteins are of the G0 glycanspecies.

Methods for monitoring changes in the N-glycosylation pattern ofglycoproteins, also referred to as glycosylation profiles, are wellknown in the art and include, but are not limited to, matrix-assistedlaser desorption ionization time-of-flight (MALDI-TOF) massspectrometry, for example, using the modified MALDI-TOF assay disclosedin Example 3 herein below, liquid chromatograph mass spectrometry(LC-MS), gas chromatography, anion-exchange chromatography,size-exclusion chromatography, high-concentration polyacrylamide gelelectrophoresis, nuclear magnetic resonance spectroscopy, and capillaryelectrophoresis and capillary gel electrophoresis, fluorescence labelingand detection by high-performance liquid chromatography (HPLC) and QTOF,and the like. In this manner, changes in the N-glycosylation pattern dueto inhibition of the expression or function of XylT and/or FucT in astably transformed plant of the invention can be monitored by subjectinga sample (for example, a leaf tissue sample) obtained from the stablytransformed plant to total N-glycan analysis by MALDI-TOF massspectrometry, and comparing the results with those obtained for acomparable sample from a control plant, wherein the control plant hasnot been genetically modified to inhibit expression or function of XylTand/or FucT. A reduction in the amount of xylose and/or fucose residuesin the N-glycans can be monitored by a reduction of the mass of therespective peaks. See, for example, Strasser et al. (2004) FEBS Letters561:132-136.

Similarly, the glycosylation profile of any given recombinantly producedglycoprotein is readily determined using standard techniques well knownto those in the art. See, for example, the review provided in Morelleand Michalski (2005) Curr. Anal. Chem. 1:29-57; herein incorporated byreference in its entirety. Thus, glycoproteins that have beenrecombinantly produced in a host organism, including a plant host, canbe analyzed for the ratio of the particular N-linked glycan structuresattached thereto. In this manner, a sample comprising isolatedrecombinantly produced glycoprotein can be subjected to enzymatic orchemical reaction to release the individual glycan structures from theglycoprotein. Following this deglycosylation step, analysis of theglycosylation profile can be carried out using any of the analyticalassays described herein above.

Recombinantly produced glycoprotein products typically exist as adiverse population of glycoforms carrying between one and several dozendifferent glycans in variable molar amounts at glycosylation sites withvarying degrees of site occupancy. Depending upon the glycoprotein,different glycoforms can yield different functional profiles. Thus, insome embodiments of the invention, it is desirable to determine theglycosylation profile of the glycoprotein having the N-glycans intact.Any technique known in the art for determining the glycosylation profileof an intact glycoprotein can be used, including the mass spectrometrymethods noted above and in the examples herein below.

By reducing or eliminating the expression or function offucosyltransferase and/or xylosyltransferase in the manner set forthherein, either transiently or stably, it is possible to produce atransgenic higher plant having the ability to produce glycoproteinshaving an N-glycosylation profile with reduced heterogeneity relative tothat normally observed for glycoproteins produced by this plant whenexpression or function of these enzymes has not been altered (i.e., theplant has the native or wild-type glycosylation machinery). Whereexpression or function of one or both of these enzymes is stably reducedor eliminated using one or more of the methods described herein above,the reduction in the heterogeneity of the N-glycosylation profile ofglycoproteins produced by the transgenic plant can be maintained fromplant generation to plant generation, including with asexual or sexualreproduction, and can be maintained across cultural conditions and withscale-up in production.

In this manner, the present invention provides a method for reducingheterogeneity of the N-glycosylation profile of a glycoprotein producedin a higher plant, for example, a dicotyledonous or monocotyledonousplant, for example, a duckweed plant. The method comprises introducinginto the plant a nucleotide construct described herein such that theexpression or function of fucosyltransferase and/or xylosyltransferaseis reduced or eliminated within the plant. In some embodiments of theinvention, the method for reducing heterogeneity of the N-glycosylationprofile of a glycoprotein produced in a higher plant comprisesintroducing into the higher plant of interest at least one nucleotideconstruct described herein above, where the nucleotide construct(s)provides for suppression of the expression of fucosyltransferase and/orxylosyltransferase in the plant, for example, using one or more of themethods described herein above.

By “reducing heterogeneity of the N-glycosylation profile” it isintended that the N-glycosylation profile is characterized by areduction in the total number of distinct N-glycan species that appearin the profile. Thus, for example, where a glycoprotein produced by ahigher plant having the native or wild-type glycosylation machinery (andthus which has not been genetically modified to reduce or eliminateexpression of fucosyltransferase and xylosyltransferase) produces aglycoprotein with an N-glycosylation profile characterized by thepresence of a mixture of 5 N-glycan species, the methods of theinvention can be used to reduce the number of N-glycan species appearingin the N-glycosylation profile. In this manner, when that higher plantis genetically modified in the manner set forth herein to reduce oreliminate expression or function of fucosyltransferase and/orxylosyltransferase, the N-glycosylation profile of this glycoproteinwould be characterized by a reduction in the number of N-glycan speciesappearing in the profile, for example, a mixture of fewer than 5N-glycan species, for example, 4, 3, or 2 N-glycan species, or even asingle N-glycan species. Where heterogeneity of the N-glycosylationprofile is reduced such that the profile is characterized by thepresence of a single predominant N-glycan species, the N-glycosylationprofile would be substantially homogeneous for that N-glycan species.

In some embodiments, the methods for reducing the heterogeneity of theN-glycosylation profile of a glycoprotein produced in a higher plantresult in the produced glycoprotein having an N-glycosylation profilethat is substantially homogeneous for the G0 glycan species. In suchembodiments, the methods for reducing the heterogeneity of theN-glycosylation profile of a glycoprotein produced in a higher plantresult in the produced glycoprotein having a substantially homogeneousN-glycosylation profile, wherein at least 80%, at least 85%, at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of thetotal amount of N-glycan species appearing in the N-glycosylationprofile for the glycoprotein is represented by the G0 glycan species. Inthese embodiments, a trace amount of precursor N-glycan species mayappear in the N-glycosylation profile as noted elsewhere herein, whereany given precursor N-glycan species that is present in theN-glycosylation profile is present at less than 5%, preferably less than4%, less than 3%, less than 2%, less than 1%, and even less than 0.5% oreven less than 0.1% of the total amount of N-glycan species appearing inthe profile.

The glycoprotein can be an endogenous glycoprotein of interest, or canbe a heterologous glycoprotein that is produced by the higher plant ofinterest, for example, a mammalian glycoprotein, including, for example,the glycoproteins described elsewhere herein. In some embodiments, theglycoprotein is a monoclonal antibody. In other embodiments, theglycoprotein is selected from the group consisting of an interferon,erythropoietin (EPO), tissue plasminogen activator (tPA), plasminogen,granulocyte-macrophage colony stimulating factor (GM-CSF), andtherapeutic immunoglobulins.

Using the methods of the present invention, it is possible to maintainthe reduced heterogeneity within the N-glycosylation profile for aglycoprotein produced in the transgenic plant with scale-up inproduction, and thus the plant continues to produce the glycoproteinsuch that its N-glycosylation profile is characterized by a reduction inthe number of N-glycan species appearing in the profile. By “scale-up inproduction” or “increase in production scale” is intended an increase inthe amount of plant biomass that is present within a culture system(i.e., a culture vessel or culture container within which the plant iscultured) that is being used to produce a protein of interest, in thiscase, a glycoprotein of interest. Thus, scale-up in production occurs,for example, when scaling up production from a scale that is suitablefor research purposes to one that is suitable for pilot production, andfurther up to a scale that is suitable for commercial production of theglycoprotein of interest.

In some embodiments, the transgenic higher plant is a monocotyledonousplant, for example, a duckweed plant, that serves as a host forrecombinant production of a glycoprotein, and the reduced heterogeneityof the N-glycosylation profile of the recombinantly producedglycoprotein is maintained with an increase in production scale, wherethe production scale is increased by at least 300-fold, at least500-fold, at least 700-fold, at least 1,000-fold, at least 1,500-fold orgreater over the initial starting biomass. In some of these embodiments,the transgenic higher plant is a duckweed plant that recombinantlyproduces a glycoprotein of interest, and the reduced heterogeneity ofthe N-glycosylation profile is maintained with an increase in productionscale, where the production scale is increased by at least 2,000-fold,at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least6,000-fold, at least 6,500-fold, or greater over the initial startingbiomass. In one such embodiment, the higher plant is a duckweed plantthat recombinantly produces a glycoprotein of interest, and the reducedheterogeneity of the N-glycosylation profile for that glycoprotein ismaintained with an increase in production scale, where the productionscale is increased by at least 7,000-fold, 8,000-fold, 9,000-fold,10,000-fold, 12,500-fold, 15,000-fold, 17,500-fold, 20,000-fold,23,000-fold, 26,000-fold, or greater over the initial starting biomass.

Furthermore, when the transgenic plant of interest is to be maintainedby continuous clonal culture, the resulting transgenic line continues toproduce glycoproteins that exhibit the reduced heterogeneity withintheir N-glycosylation profile. Continuous clonal culture can be achievedusing any suitable method known in the art. In some embodiments,continuous clonal culture is achieved by periodically taking one or moresubsamples of the plant culture and transferring the subsample(s) tofresh culture medium for further culture. Thus, for example, in someembodiments, the transgenic plant line that is maintained by continuousclonal culture is a duckweed transgenic plant line that has beengenetically modified to reduce or eliminate expression or function offucosyltransferase and/or xylosyltransferase. In this manner, thereduced heterogeneity of the N-glycosylation profile of glycoproteinsproduced in the transgenic plant line is maintained with continuousclonal culture of the transgenic plant line for at least 8 months, atleast 10 months, at least 1 year, at least 1.5 years, at least 2 years,at least 2.5 years, at least 3 years, at least 3.5 years, at least 4years, at least 4.5 years, at least 5 years, or longer, and can bemaintained for as long as the transgenic plant line is maintained.

Heterologous Polypeptides and Glycoproteins

Higher plants, particularly higher plants that serve as expressionsystems for recombinant proteins for pharmaceutical use, that have beenstably transformed to produce glycoproteins with an alteredN-glycosylation pattern using the methods described herein may begenetically modified to produce any recombinant protein of interest.Where the recombinant protein is one in which post-translationalglycosylation is applicable, the methods of the invention advantageouslyprovide a means to produce these glycoproteins with an N-glycosylationpattern that more closely reflects that of mammalian hosts, particularlya glycosylation pattern that is “humanized.” Examples of recombinantproteins of interest include, but are not limited to, insulin, growthhormone, plasminogen, α-interferon, β-interferon, β-glucocerebrosidase,β-glucoronidase, retinoblastoma protein, p53 protein, angiostatin,leptin, monoclonal antibodies and fragments thereof, cytokines, forexample, erythropoietin (EPO), granulocyte macrophage colony stimulatingfactor, tissue plasminogen activator, blood coagulation factors, forexample, Factor VII, Factor VIII, Factor IX, and activated protein C,alpha 1-antitrypsin, receptors, hormones, human vaccines, animalvaccines, peptides, and serum albumin. Furthermore, the transgenichigher plants of the invention are capable of producing a glycoproteinproduct that has a substantially homogenous glycosylation profile forthe G0 glycan species, and which is characterized by its substantialhomogeneity for the G0 glycoform. This advantageously results in planthost expression systems that have increased production consistency, aswell as reduced chemical, manufacturing, and control (CMC) riskassociated with the production of these glycoprotein compositions.

The glycoprotein compositions produced in accordance with the methods ofthe present invention may be contained in a composition comprising apharmaceutically acceptable carrier. Such compositions are useful in amethod of treating a subject for a disease or disorder for whichtreatment with the glycoprotein will provide a therapeutic benefit. Inthis manner, glycoproteins that have been produced in a plant, forexample, a duckweed plant, stably transformed in accordance with themethods of the present invention can be administered to a subject inneed thereof.

The glycoprotein compositions of the invention comprise N-linked glycansthat are predominately of the G0 glycan structure. In this manner, thepresent invention provides glycoprotein compositions that haveglycosylation profiles that are “substantially homogeneous” or“substantially uniform” or have “substantial homogeneity” as definedherein above. Thus, in some embodiments, the glycoprotein compositionsare substantially homogeneous for the G0 glycan species, and thus have asubstantially homogeneous glycosylation profile, wherein at least 80%,at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least 99% of the total amount of N-glycan species appearing in theglycosylation profile for the composition is represented by the G0glycan species, with a trace amount of precursor N-glycan speciesappearing in the glycosylation profile, i.e., any given precursorN-glycan species that is present in the glycosylation profile is presentat less than 5%, preferably less than 4%, less than 3%, less than 2%,less than 1%, and even less than 0.5% or even less than 0.1% of thetotal amount of N-glycan species appearing in the profile. For such acomposition, a representative precursor N-glycan species appearing inits glycosylation profile would be the Man3GlcNAc2, MGn(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,3 mannose arm),and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,6mannose arm) precursor N-glycan species described above, where anysingle one or any combination of these precursor N-glycan species can bepresent.

In this manner, the invention provides “substantially homogeneous” or“substantially uniform” glycoprotein compositions or glycoproteincompositions having “substantial homogeneity” as defined herein above.In some embodiments, the invention provides substantially homogeneousglycoprotein compositions, wherein at least 80%, at least 85%, at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of theglycoprotein present in the composition is represented by the G0glycoform, wherein all anticipated glycosylation sites are occupied bythe G0 glycan species, with a trace amount of precursor or undesiredglycoforms being present in the composition, i.e., the precursorglycoforms represent less than 5%, less than 4%, less than 3%, less than2%, less than 1%, or even less than 0.5%, or less than 0.1% of the totalglycoforms present within the composition. In such a composition, arepresentative precursor glycoform would be one in which glycosylationsites are unoccupied, and an exemplary undesired glycoform would be aglycoform having a mixture of G0 glycan and G0X or G0XF3 glycan speciesattached to its glycosylation sites.

In some embodiments of the invention, the plant host comprises one ormore polynucleotides that provide for expression of an antibody thatspecifically binds to a mammalian protein of interest, particularly ahuman protein of interest. Thus, in one aspect, the invention providesmethods for producing monoclonal antibodies in higher plants, whereinthe monoclonal antibodies have an N-glycosylation pattern that reflectsa reduction in the amount of β1,2-linked xylose residues and α1,3-linkedfucose residues within the N-linked glycans, and compositions comprisingrecombinant monoclonal antibodies produced using plant hosts geneticallymodified in the manner set forth herein. In some embodiments, the planthost of interest is a member of the duckweed family.

Monoclonal antibodies are increasingly being used as therapeutic agentsto treat human disease, including, but not limited to, cancer anddiseases having an autoimmune or inflammatory component. See, forexample, King (1999) Curr. Opin. Drug Discovery Dev. 2:110-17; Vaswaniand Hamilton (1998) Ann. Allergy Asthma Immunol. 81:105-19; and Holligerand Hoogenboom, Nat. Biotechnology 16:1015-16; each of which is hereinincorporated by reference. Although some of these antibodies havetherapeutic effects that result solely from antigen binding, for exampleantibodies that bind to a receptor or ligand to prevent ligand-receptorinteractions, other antibodies need effector functions such as therecruitment of the immune system to kill target cells in order to betherapeutically active. See, for example, Clynes et al. (2000) Nat. Med.6:443-46; Clynes et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:652-56,and Anderson et al. (1997) Biochem. Soc. Trans. 25:705-8; each of whichis herein incorporated by reference.

The antigen-recognition activities and effector functions of antibodiesreside in different portions of the antibody molecule. The Fab′ portionof the antibody provides antigen recognition activity, while the Fcportion provides effector functions such as the activation of accessoryeffector cells including phagocytic cells (macrophages and neutrophils),natural killer cells, and mast cells. Antibodies bind to cells via theFc region, with an Fc receptor site on the antibody Fc region binding toan Fc receptor (FcR) on a cell. There are a number of Fc receptors thatare specific for different classes of antibodies, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), initiation of complement-dependent cytotoxicity (CDC), release ofinflammatory mediators, and control of immunoglobulin production.Methods for assaying effector function of antibodies are well known inthe art and include those assaying for CDC, ADCC, and apoptosis. See,for example, Subbramanian et al. (2002) J. Clin. Microbiol.40:2141-2146; Ahman et al. (1994) J. Immunol. Methods 36:243-254;Brezicka et al. (2000) Cancer Immunol. Immunother. 49:235-242;Gazzano-Santoro et al. (1997) J. Immunol. Methods 202:163-171; Prang etal. (2005) British J. Cancer 92:342-349; Shan et al. (1998) Blood92:3756-3771; Ghetie et al. (2001) Blood 97:1392-1398; and, Mathas etal. (2000) Cancer Research 60:7170-7176; all of which are hereinincorporated by reference.

It is known in the art that the glycosylation status of the Fc portionof an antibody molecule plays a key role in determining whether anantibody will have effector function. See, for example, Tao and Morrison(1987) J. Immunol. 143:2595-601; Wright and Morrison (1997) Trends inBiotech. 15:26-32; Wright and Morrison (1998) J. Immunol. 160:3393-402;Mimura et al. (2000) Mol. Immunol. 37:697-706; Jefferis and Lund (2002)Immunol. Lett. 82:57-65; Krapp et al. (2003) J. Mol. Biol. 325:979-89;and Jefferis (2005) Biotechnol. Prog. 21:11-16; each of which is hereinincorporated by reference. Glycosylation of recombinantly producedantibodies varies depending on the expression system used. See, forexample, Raju et al. (2000) Glycobiology 10:477-86; Wright and Morrison(1997) Trends in Biotech. 15:26-32. Further, where the N-glycosylationpattern of a mammalian-produced monoclonal antibody is altered to reduceor deplete the α(1,6)-linked core fucose residue, the monoclonalantibody exhibits increased effector function, particularly increasedADCC activity. See, for example, U.S. Pat. No. 6,946,292. For somemammalian-produced monoclonal antibodies, where the N-glycosylationpattern is altered to reduce or deplete the β(1,4)-galactose residuesattached to the 1,3 and/or 1,6 mannose arms, activation ofcomplement-dependent cytotoxicity (CDC) against antigen-bearing targetcells may be reduced without altering other functional activities of theantibody, including ADCC activity. See, for example, Boyd et al. (1995)Mol. Immunol. 32:1311-1318.

Antibodies having antigen recognition activity, and in some embodimentsimproved effector function, may be produced by a higher plant host, suchas duckweed, that has been stably transformed in the manner set forthherein to alter its glycosylation machinery. Accordingly, the presentinvention provides methods for producing a recombinant monoclonalantibody, including a monoclonal antibody having improved effectorfunction, wherein the antibody is recombinantly produced within a planthaving an altered N-glycosylation pattern of endogenous and heterologousglycoproteins produced therein such that these glycoproteins exhibit areduction in the amount of the plant-specific β1,2-linked xyloseresidues and/or α1,3-linked fucose residues attached to the N-glycansthereof. Where the antibodies have reduced amounts α1,3-linked fucoseresidues attached to the N-glycans thereof, the antibodies may haveincreased ADCC activity relative to antibodies produced in a controlplant that has not been genetically modified to inhibit expression orfunction of FucT.

Also encompassed are recombinant monoclonal antibodies having effectorfunction, and in some embodiments, improved effector function, where theantibodies are produced in a duckweed expression system that has beengenetically modified to inhibit expression or function of the FucT ofSEQ ID NO:3 and/or the XylT of SEQ ID NO:6, and any isoforms thereof,for example, the XylT isoform #2 comprising the sequence set forth inSEQ ID NO:21 (encoded by SEQ ID NO:20). Thus, in some embodiments, theplant serving as the host for recombinant production of the monoclonalantibody is a member of the Lemnaceae as noted elsewhere herein, forexample, a Lemna plant, comprising, for example, a XylT RNAi expressioncassette and/or a FucT RNAi expression cassette described above stablyintegrated within its genome. In this manner, the present inventionprovides a method for producing a recombinant monoclonal antibody havingan N-glycosylation pattern that more closely resembles that found in amammalian host expression system, and with improved effector function,where the method comprises expressing one or more chains of the antibodyin a duckweed plant, or duckweed cell or duckweed nodule, that has beengenetically modified to alter the glycosylation machinery such that therecombinantly produced monoclonal antibody exhibits a reduction in theattachment of the plant β1,2-linked xylose residue and/or α1,3-linkedfucose residue to the N-glycans thereof, and culturing the duckweedplant, or duckweed cell or duckweed nodule, under conditions suitablefor expression of the monoclonal antibody.

Thus the present invention provides novel antibody compositions whereinthe antibody comprises N-linked glycans that are predominately of the G0glycan structure. In this manner, the present invention providesantibody compositions, for example, monoclonal antibody compositions,that have glycosylation profiles that are “substantially homogeneous” or“substantially uniform” or have “substantial homogeneity” as definedherein above. Thus, in some embodiments, the antibody compositions aresubstantially homogeneous for the G0 glycan species, and thus have asubstantially homogeneous glycosylation profile, wherein at least 80%,at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least 99% of the total amount of N-glycan species appearing in theglycosylation profile for the composition is represented by the G0glycan species, with a trace amount of precursor N-glycan speciesappearing in the glycosylation profile, i.e., any given precursorN-glycan species that is present in the glycosylation profile is presentat less than 5%, preferably less than 4%, less than 3%, less than 2%,less than 1%, and even less than 0.5% or even less than 0.1% of thetotal amount of N-glycan species appearing in the profile. For such acomposition, a representative precursor N-glycan species appearing inits glycosylation profile would be, for example, the Man3GlcNAc2, MGn(GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,3 mannose arm),and GnM (GlcNac1Man3GlcNAc2 wherein GlcNac1 is attached to the 1,6mannose arm) precursor N-glycan species described above, where anysingle one or any combination of these precursor N-glycan species can bepresent.

In one such embodiment, the monoclonal antibody composition has asubstantially homogeneous glycosylation profile, wherein 95.8% of thetotal amount of N-glycan species appearing in the glycosylation profilefor the composition is represented by the G0 glycan species(GlcNAc₂Man₃GlcNAc₂), with the following precursor N-glycan speciesappearing in the glycosylation profile: Man₃GlcNAc₂ (0.67%),GlcNAcMan₃GlcNAc₂ (1.6%), GalGlcNAc₂Man₃GlcNAc₂ (1.2%), Man₆GlcNAc₂(0.21%), Man₇GlcNAc₂ (0.30%), and Man₈GlcNAc₂ (0.28%). This can becompared with the monoclonal antibody composition obtained from the“wild-type” duckweed plant expression system wherein the same monoclonalantibody is expressed but where the glycosylation machinery of theduckweed plant has not been genetically modified to inhibit expressionof XylT and FucT. Such a “wild-type”-derived monoclonal antibodycomposition has a more heterogeneous glycosylation profile that ischaracterized by two predominant N-glycan species, i.e., G0XF³ and G0X,with several precursor N-glycan species represented in trace amounts. Inone such embodiment, the “wild-type”-derived monoclonal antibodycomposition has a glycosylation profile with the following N-glycanspecies represented therein: G0 (GlcNAc₂Man₃GlcNAc₂) (8.4%); G0X(GlcNAc₂[Xyl]Man₃GlcNAc₂) (17.2%); G0XF³ (GlcNAc₂[Xyl]Man₃[Fuc]GlcNAc₂)(67.4%); Man₃GlcNAc₂ (0.26%); GlcNAcMan₃GlcNAc₂ (0.40%);(Xyl)Man₃(Fuc)GlcNAc₂ (0.76%); GlcNAc₂Man₃(Fuc)GlcNAc₂ (2.1%);GlcNAc(Xyl)Man₃(Fuc)GlcNAc₂ (1.4%); Man₆GlcNAc₂ (0.21%); Man₇GlcNAc₂(0.63%); Gal(Fuc)GlcNAc₂(Xyl)Man₃(Fuc)GlcNAc₂ (0.26%); Man₈GlcNAc₂(0.61%); and Man₉GlcNAc₂ (0.40%).

In this manner, the invention provides “substantially homogeneous” or“substantially uniform” antibody compositions or antibody compositionshaving “substantial homogeneity” as defined herein above. In someembodiments, the invention provides substantially homogeneous antibodycompositions, for example, monoclonal antibody compositions, wherein atleast 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99% of the antibody present in the composition isrepresented by the G0 glycoform, wherein all anticipated glycosylationsites (for example, each of the Asn-297 residues of the C_(H)2 domainsof the heavy chains of an IgG-type antibody) are occupied by the G0glycan species, with a trace amount of precursor glycoforms beingpresent in the composition. In one such composition, the precursorglycoforms are selected from the group consisting of an antibody havingan Fc region wherein the C_(H)2 domain of one heavy chain has a G0glycan species attached to Asn 297, and the C_(H)2 domain of the otherheavy chain is unglycosylated; an antibody having an Fc region whereinthe C_(H)2 domain of one heavy chain has a G0 glycan species attached toAsn 297, and the C_(H)2 domain of the other heavy chain has the GnM orMGn precursor glycan attached to Asn 297; and an antibody having an Fcregion wherein the Asn 297 glycosylation site on each of the C_(H)2domains has a G0 glycan species attached, with a third G0 glycan speciesattached to an additional glycosylation site within the mAb structure;wherein a trace amount of these precursor glycoforms is present, i.e.,the precursor glycoforms represent less than 5%, less than 4%, less than3%, less than 2%, less than 1%, or even less than 0.5%, or less than0.1% of the total glycoforms present within the antibody composition.

The substantially homogeneous antibody compositions of the inventionwherein at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or at least 99% of the antibody present in thecomposition is represented by the G0 glycoform represent“glycan-optimized antibodies.” By “glycan-optimized antibodies” isintended the antibodies of the invention have been geneticallyengineered in their glycosylation pattern such that they aresubstantially homogeneous for the G0 glycoform, which yields an antibodyhaving improved Fc effector function. By “improved Fc effector function”is intended these antibodies have increased ADCC activity relative tosame-sequence antibodies (i.e., antibodies that have the same amino acidsequence) that, as a result of the production process, have a moreheterogeneous glycosylation profile. Thus, for example, antibodiesproduced in mammalian host expression systems, for example CHO cells, ininsect host cells, in yeast cells, or in other plant host expressionsystems that have not been genetically altered to inhibit XylT and FucTexpression tend to have more heterogeneous glycosylation profiles, andthus a mixture of glycoforms, that can effect overall effector functionof the antibody product. The G0 glycoform of the antibody compositionsof the present invention advantageously provides an antibody compositionthat has increased ADCC activity in association with the absence offucose residues. In some embodiments, ADCC activity is increased by25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold,300-fold, 400-fold, 500-fold, or even 1000-fold relative tosame-sequence antibodies having a heterogeneous glycosylation profile(i.e., with multiple glycoforms present as major glycoforms in theantibody composition). Furthermore, the G0 glycoform lacks the terminalGal residues present in antibodies having the G2 glycoform. As such,these substantially homogeneous antibody compositions of the inventionhaving predominately the G0 glycoform have increased ADCC/CDC ratios. Inaddition, the substantially homogeneous antibody compositions of theinvention having predominately the G0 glycoform have increased bindingto the FcγRIII, for example, FcγRIIIa, wherein binding affinity isincreased about 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, up to100-fold over that observed for same-sequence antibody compositionshaving a heterogeneous glycosylation profile, and thus a mixture ofglycoforms. For oncology and autoimmune diseases, therapeutic antibodieshaving increased binding affinity for Fc receptors, for example,FcγRIII, has been strongly correlated with increased efficacy andimproved response to treatment.

In some embodiments of the invention, the substantially homogeneousantibody compositions of the invention having predominately the G0glycoform have altered CDC activity when compared to that observed forsame-sequence antibody compositions having a heterogeneous glycosylationprofile, and thus a mixture of glycoforms. For example, in one suchembodiment, the substantially homogeneous antibody compositions of theinvention have predominately the G0 glycoform and have decreased CDCactivity when compared to that observed for same-sequence antibodycompositions having a heterogeneous glycosylation profile, and thus amixture of glycoforms. Thus, in some embodiments, the present inventionprovides substantially homogeneous antibody compositions havingpredominately the G0 glycoform and CDC activity that is decreased by asmuch as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or even 100% when compared tosame-sequence antibody compositions having a heterogeneous glycosylationprofile. In some of these embodiments, the substantially homogeneousantibody compositions having predominately the G0 glycoform anddecreased CDC activity are further characterized by having increasedADCC activity when compared to same-sequence antibody compositionshaving a heterogeneous glycosylation profile.

Without being bound by any theory or mechanism of action, asubstantially homogeneous G0 glycoform antibody composition of thepresent invention having decreased CDC activity, and similar orincreased ADCC activity in the manner described above, mayadvantageously provide for increased cytotoxicity against target cellswhile reducing potential adverse side effects that may be associatedwith complement activation following its administration. By reducing thepotential for these adverse side effects, the substantially homogeneousantibody compositions having predominately the G0 glycoform canadvantageously be administered at faster infusion rates, therebyreducing dosing time at any given administration, and/or can be dosed athigher initial concentrations if warranted, with reduced concern fortriggering adverse side affects associated with complement activation.

For example, complement activation plays a pivotal role in thepathogenesis of moderate to severe first-dose side effects of treatmentwith the chimeric anti-CD20 monoclonal antibody IDEC-C2B8 (IDECPharmaceuticals Corp., San Diego, Calif.; commercially available underthe tradename Rituxan®, also referred to as rituximab). See, forexample, van der Kolk et al. (2001) British J. Haematol. 115:807-811.The rituximab antibody within the Rituxan® product is expressed withinChinese hamster ovary (CHO) cells, and thus the antibody compositioncomprises a heterogeneous glycosylation profile (i.e., a mixture ofglycoforms). CDC activity of rituximab has been shown to be correlatedwith galactose content. In this manner, as the number of galactoseresidues increases from 0-2 moles/mole of heavy chain, the level of CDCactivity increases from 80% (β-galactosidase treated to remove allβ(1,4)-galactose residues from the 1,3 and 1,6 mannose arms of theN-glycans attached to Asn 297 of the C_(H)2 domains of the heavy chains)to 150% (UDP galactosyl transferase treated to ensure β(1,4)-galactoseresidues are attached to both the 1,3 and 1,6 mannose arms of theN-glycans attached to Asn 297 sites) of the maximum observed for theantibody having 1 mole galactose/mole of heavy chain (see, IDEC BLA97-0260 at the website fda.gov/Cder/biologics/review/ritugen112697,available on the worldwide web). A substantially homogeneous anti-CD20antibody composition comprising anti-CD20 antibody having the samesequence as rituximab and having predominately the G0 glycoform wouldadvantageously have decreased CDC activity, thereby reducing thepotential for adverse side effects normally associated with complementactivation upon antibody administration when the antibody compositioncomprises a heterogenous glycosylation profile (i.e., a mixture ofglycoforms).

Thus, a substantially homogeneous G0 glycoform antibody composition ofthe present invention having decreased CDC activity, and the same orincreased ADCC activity, can advantageously be used in therapeuticapplications that have heretofore been unsuitable, inadvisable, orinefficacious for one or more patient populations as a result ofcomplications due to adverse side effects normally associated withcomplement activation upon administration of the same-sequence antibodycomposition that comprises a heterogeneous glycosylation profile (i.e.,a mixture of glycoforms). Such side effects include, but are not limitedto, moderate to severe side effects that can be associated withfirst-time and/or rapid administration of an antibody, including, forexample, fever and/or chills, nausea, dyspnea, flushes, and the like.See, for example, van der Kolk et al. (2001) British J. Haematol.115:807-811 and the references cited therein; Winkler et al. (1999)Blood 94:2217-2224). In this manner, the present invention provides amethod for reducing one or more adverse side effects related tocomplement activation upon administration of an antibody, for example, amonoclonal antibody, the method comprising administering the antibody asa substantially homogeneous antibody composition as defined hereinabove, and thus at least 80%, at least 85%, at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least 99% of the antibody present in thecomposition is represented by the G0 glycoform, with a trace amount ofprecursor glycoforms being present in the composition. In someembodiments, at least 90%, at least 95%, or at least 99% of the antibodypresent in the composition is represented by the G0 glycoform, with atrace amount of precursor glycoforms being present in the composition.

As a result of their increased Fc effector function, the substantiallyhomogenous antibody compositions of the invention having thepredominately G0 glycoform provide the opportunities for new andimproved routes of administration, for example, extending the possibleroutes of administration for known therapeutic antibodies to otherroutes of administration beyond infusion and intravenous administration,for example, to subcutaneous administration. Furthermore, as a result oftheir increased potency, the antibody compositions of the invention canbe dosed at lower concentrations, or dosed at lower volumes, and dosedwith less frequency. A reduction in the volume of the administeredantibody composition is particularly advantageous in those instanceswhere adverse events resulting from infusion reactions with a monoclonalantibody are volume-related. The increased potency of the antibodycompositions of the invention also opens up new clinical indications forexisting mAb targets that may not have been responsive to antibodytherapy with more heterogenous glycoform antibody compositions, andprovides the ability to revisit previously undeveloped mAb targets.

The monoclonal antibodies produced in accordance with the methods of thepresent invention may be contained in a composition comprising apharmaceutically acceptable carrier. Such compositions are useful in amethod of treating a subject in need of an antibody having effectorfunction, and in some embodiments, improved effector function where FucTexpression has been targeted for inhibition. In this manner, monoclonalantibodies produced in a plant, for example, a duckweed plant, stablytransformed in accordance with the methods of the present invention canbe administered to a subject in need thereof.

Any antibody may be produced within a plant host genetically modifiedaccording to the methods of the invention. In one embodiment, theantibody is a therapeutic antibody. Antibodies have been approved forthe treatment of a number of disorders, and many additional therapeuticantibodies are in development. See, Brekke and Sandlie (2003) Nat. Rev.Drug. Discov. 2(3):240, herein incorporated by reference. Examples oftherapeutic antibodies that may be expressed by the methods of theinvention include, but are not limited to, anti-CD3 antibodies targetingthe CD3 antigen (e.g., OKT®3); anti-platelet gpIIb/IIIa antibodies (e.g.abciximab (previously known as c7E3 Fab), distributed as ReoPro®);anti-EpCAM antibodies (e.g., Panorex®); anti-CD20 antibodies targetingthe CD20 antigen (e.g. rituximab, distributed as Rituxan®; see U.S. Pat.No. 5,736,137, herein incorporated by reference); anti IL-2 receptor(e.g. Zenapax®, Simulect®); anti-ERBB2 (e.g. trastuzumab, distributed asHerceptin®; see U.S. Pat. No. 6,165,464, herein incorporated byreference); anti-TNF-α (e.g., infliximab, distributed as Remicade®),anti-F-protein (e.g. Synagis®); anti-CD30 antibodies targeting the CD30antigen (see, for example, the 5F11 antibody described in Borchmann etal. (2003) Blood 102:3737-3742 and in Example 6 herein below, see alsoWO 03/059282 and U.S. Patent Application Publication No. 2004/0006215,herein incorporated by reference; the SGN-30 antibody described in Wahlet al. (2002) Cancer Res. 62:3736-3742, a chimeric version of the AC10anti-CD30 antibody, see also U.S. Patent Application Publication No.20040018194 and U.S. Pat. No. 7,090,843, herein incorporated byreference); anti-CD33 antibodies targeting the CD33 antigen (e.g.Mylotarg®; see U.S. Pat. No. 5,733,001, herein incorporated byreference); anti-CD52 antibodies targeting the CD52 antigen (e.g.,alemtuzumab, distributed as Campath®; see U.S. Pat. No. 5,846,534,herein incorporated by reference); anti-CD20 (e.g., Zevalin®); anti-IgEFc (e.g., omalizumab, marketed under Xolair®); anti-VEGF (e.g.,bevacizumab, distributed as Avastin®; see, Presta et al. (1997) CancerRes. 57:4593-9; herein incorporated by reference); anti-EGF-R (e.g.,cetuximab, distributed as Erbitux®)); anti-CD11a (e.g., efalizumab,distributed under Raptiva®), anti-IL-8; anti-C5; anti-TNF-α (e.g.,adalimumab, distributed as Humira®); anti-TGF-β2; anti-IL2 receptor(e.g., daclizumab (Zenapax) and basiliximab (Simulect));anti-α-4-integrin; anti-CD4; anti-CD2; anti-CD19 (e.g., MT103, abispecific antibody); anti-CD22 antibody targeting the CD22 antigen(e.g., the monoclonal antibody BL-22); anti-CD23 antibody targeting theCD23 antigen on malignant B cells (e.g., IDEC-152); anti-CD80 antibodytargeting the CD80 antigen (e.g., IDEC-114); anti-CD38 antibodytargeting the CD38 antigen on malignant B cells; α-M-CSF antibodytargeting macrophage colony stimulating factor; antibodies targeting thereceptor activator of nuclear factor-kappaB (RANK) and its ligand(RANKL), which are overexpressed in multiple myeloma; anti-CD40antibodies (e.g., SGN-40) targeting the CD40 antigen on malignant Bcells; antibodies targeting tumor necrosis factor-relatedapoptosis-inducing ligand receptor 1 (TRAIL-R1) (e.g., the agonistichuman monoclonal antibody HGS-ETR1) and TRAIL-R2 expressed on a numberof solid tumors and tumors of hematopoietic origin) anti-HBV antibodies;or anti-MHC class II antibodies. Many of these antibodies are known torequire effector activity for function, either CDC and/or ADCC, forexample, Herceptin®, Rituxan®, Mylotarg®, and Campath®.

In some embodiments, the plant host that has been genetically modifiedto alter its glycosylation machinery in the manner set forth herein, forexample, duckweed, may express biologically active α-2b-interferon, forexample human α-2b-interferon precursor (NCBI Protein Accession No.AAB59402) or mature human α-2b-interferon (amino acids 24-188 of NCBIProtein Accession No. AAB9402) or biologically active variants thereof.Examples of biologically active variants of human α-2b-interferon areknown in the art. See, for example, WO 2005/035767, disclosing truncatedversions of α-2b-interferon; European patent EP211148B1; and U.S. Pat.Nos. 4,748,233, 4,801,685, 4,816,566, 4,973,479, 4,975,276, 5,089,400,5,098,703, 5,231,176, and 5,869,293; herein incorporated by reference.In other embodiments, the plant host, for example, duckweed, may expressbiologically active mature human growth hormone with or without itsaccompanying signal peptide. The plant host may also expressbiologically active variants of human growth hormone. See, for example,WO 02/10414, directed to expression of biologically active polypeptidesusing a duckweed expression system.

In yet other embodiments, the plant host, for example, duckweed, mayexpress plasminogen, microplasminogen, or biologically active variantsthereof. The plasminogen or microplasminogen to be expressed in theplant host may be from any mammalian source. In some embodiments, theplasminogen or microplasminogen is human or porcine. See, for example,WO 2005/078109, directed to expression of these proteins using aduckweed expression system.

By “biologically active variant” of these heterologous polypeptides isintended a polypeptide derived from the native polypeptide by deletion(so-called truncation) or addition of one or more amino acids to theN-terminal and/or C-terminal end of the native protein; deletion oraddition of one or more amino acids at one or more sites in the nativeprotein; or substitution of one or more amino acids at one or more sitesin the native protein. Variants of a heterologous polypeptide ofinterest encompassed by the present invention retain the biologicalactivity of the native polypeptides.

For example, biologically active variants of α-2b-interferon continue topossess the desired biological activity of the native α-2b-interferonincluding the ability to increase resistance to viral infection, or theability to modulate the transcription of α-2b-interferon-regulated genetargets. Such biologically active variants may result from, for example,genetic polymorphism or from human manipulation. Biologically activevariants of a heterologous protein of interest will have at least about50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferablyat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and morepreferably at least about 98%, 99% or more sequence identity to theamino acid sequence for the heterologous protein of interest. Thus, forexample, a biologically active variant of native α-2b-interferon willhave at least about 50%, 60%, 65%, 70%, generally at least about 75%,80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, and more preferably at least about 98%, 99% or more sequenceidentity to the amino acid sequence for human α-2b-interferon precursor(NCBI Protein Accession No. AAB59402) or mature human α-2b-interferon(amino acids 24-188 of NCBI Protein Accession No. AAB9402), wheresequence identity is determined using the parameters identified hereinabove. Thus, a biologically active variant of a native heterologousprotein of interest may differ from that protein by as few as 1-15 aminoacid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4,3, 2, or even 1 amino acid residue.

In one embodiment, the plant host that has been genetically modified toalter its glycosylation machinery in the manner set forth herein is astably transformed duckweed plant or duckweed cell or duckweed nodulethat expresses biologically active polypeptides that cannot effectivelybe commercially produced by existing gene expression systems, because ofcost or logistical constraints, or both. For example, some proteinscannot be expressed in mammalian systems because the protein interfereswith cell viability, cell proliferation, cellular differentiation, orprotein assembly in mammalian cells. Such proteins include, but are notlimited to, retinoblastoma protein, p53, angiostatin, and leptin. Thepresent invention can be advantageously employed to produce mammalianregulatory proteins; it is unlikely given the large evolutionarydistance between higher plants and mammals that these proteins willinterfere with regulatory processes in duckweed. Transgenic duckweed canalso be used to produce large quantities of proteins such as serumalbumin (in particular, human serum albumin), hemoglobin, and collagen,which challenge the production capabilities of existing expressionsystems.

Finally, higher plant systems can be engineered to produce biologicallyactive multimeric proteins (e.g., monoclonal antibodies, hemoglobin,P450 oxidase, and collagen, and the like) far more easily than canmammalian systems. One exemplary approach for producing biologicallyactive multimeric proteins in duckweed uses an expression vectorcontaining the genes encoding all of the polypeptide subunits. See,e.g., During et al. (1990) Plant Mol. Biol. 15:281 and van Engelen etal. (1994) Plant Mol. Biol. 26:1701. The expression cassette comprisingthe XylT and/or FucT inhibitory polynucleotide can be introduced intosuch a vector. This vector is then introduced into duckweed cells usingany known transformation method, such as a ballistic bombardment orAgrobacterium-mediated transformation. This method results in clonalcell lines that express all of the polypeptides necessary to assemblethe multimeric protein, as well as the XylT and/or FucT inhibitorysequences that alter the glycosylation pattern of the N-glycans ofglycoproteins. Accordingly, in some embodiments, the transformedduckweed contains one or more expression vectors encoding a heavy andlight chain of a monoclonal antibody or Fab′ antibody fragment, and anexpression cassette comprising the XylT and/or FucT inhibitorypolynucleotide, and the monoclonal antibody or antibody fragment isassembled in the duckweed plant from the expressed heavy and lightchain.

A variation on this approach is to make single gene constructs, mix DNAfrom these constructs together, then deliver this mixture of DNAs intoplant cells using ballistic bombardment or Agrobacterium-mediatedtransformation. As a further variation, some or all of the vectors mayencode more than one subunit of the multimeric protein (i.e., so thatthere are fewer duckweed clones to be crossed than the number ofsubunits in the multimeric protein). In an alternative embodiment, eachduckweed clone has been genetically modified to alter its glycosylationmachinery and expresses at least one of the subunits of the multimericprotein, and duckweed clones secreting each subunit are culturedtogether and the multimeric protein is assembled in the media from thevarious secreted subunits. In some instances, it may be desirable toproduce less than all of the subunits of a multimeric protein, or even asingle protein subunit, in a transformed duckweed plant or duckweednodule culture, e.g., for industrial or chemical processes or fordiagnostic, therapeutic, or vaccination purposes.

In some embodiments of the invention, the transgenic plant host ofinterest is a “high expressor” of a glycoprotein described herein,including, for example, the glycoproteins comprising N-linked glycansthat are predominately of the G0 glycan structure. By “high expressor”is intended the transgenic plant host that has been engineered toproduce the glycoproteins described herein is capable of producing theglycoprotein of interest at a level such that the glycoprotein ofinterest represents at least 5% or more of the total soluble proteinproduced in the transgenic plant host. In some embodiments, a “highexpressor” is a transgenic plant host that has been engineered toproduce the glycoproteins described herein such that the glycoprotein ofinterest represents at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, or more of the total soluble protein produced in thetransgenic plant host. Thus, for example, in one embodiment, thetransgenic plant host is a duckweed that has been modified to inhibitexpression of XylT and FucT, and the transgenic duckweed is a highexpresser of a glycoprotein described herein. In some of theseembodiments, the transgenic duckweed is a high expressor of aglycan-optimized monoclonal antibody having the predominate G0 glycoformdescribed herein above. In yet other embodiments, the transgenicduckweed expresses the glycan-optimized monoclonal antibody such thatthis glycoprotein represents about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, or greater, of the total soluble protein.

Further Humanization of Glycoproteins

In some embodiments, it may be desirable for the glycoproteins of thepresent invention to comprise complex N-glycans having the terminalβ(1,4)-galactose residues attached to the 1,3 and/or 1,6 mannose arms.Without being bound by theory, these terminal galactose residues maycontribute to the therapeutic function and/or pharmacokinetic activityof a glycoprotein. It is recognized that the methods of the presentinvention can be paired with other methods known in the art to furthermodify the glycoproteins of the invention such that one or more of theN-glycans attached thereto comprises one or more terminal galactoseresidues, i.e., wherein one or more of the N-glycans is represented bythe G1 or G2 glycan species. In this manner, the glycoproteincompositions of the present invention, including the monoclonalantibodies described herein, can be modified, for example, enzymaticallywith use of a glycosyltransferase enzyme to obtain glycoproteins havinga substantially homogenous glycosylation profile for the G1, preferablyfor the G2 glycan species. See, for example, U.S. Patent ApplicationPublication No. 2004/0191256, herein incorporated by reference in itsentirety, teaching galactosyltransferase modification of a substrateglycoprotein to obtain a glycoprotein wherein substantially all of theN-linked glycan species are of the G2 form. In this manner, theglycoprotein of interest can be reacted with an activated galactose inthe presence of a galactosyltransferase and a metal salt. Thegalactosyltransferase can be a mammalian β1,4 galactosyltransferase(GalT), for example, human GalT, and the activated galactose can be, forexample, UDP-galactose.

Alternatively, the transgenic plants of the invention having FucT andXylT expression silenced in the manner set forth herein can be furthermodified in their glycosylation machinery such that they express agalactosyltransferase and efficiently attach the terminal galactoseresidue to the N-glycans of endogenous and heterologous glycoproteinsproduced therein. In this manner, the transgenic plants of the inventioncan be further modified by introducing a nucleotide construct, forexample, an expression cassette, that provides for the expression of agalactosyltransferase. The galactosyltransferase can be a mammalian β1,4galactosyltransferase (GalT), for example, human GalT (see, for example,U.S. Pat. No. 6,998,267, herein incorporated by reference in itsentirety) or a hybrid GalT (see, for example, WO 03/078637, hereinincorporated by reference in its entirety) comprising at least a portionof a cytoplasmic tail-transmembrane-stem region of a firstglycosyltransferase (e.g., a plant glycosyltransferase such asxylosyltransferase, N-acetylglycosaminlytransferase orfucosyltransferase) and at least a portion of a catalytic region of asecond glycosyltransefersae (e.g., mammalian glycosyltransferase, forexample, human GalT). By silencing expression of XylT and FucT in aplant, for example, a duckweed, and providing for expression of GalT,for example, human GalT, or a hybrid enzyme comprising a portion of thecatalytic domain of GalT, for example, human GalT, in this plant, forexample, duckweed, it is possible to obtain transgenic plants producingglycoproteins, both endogenous and heterologous, that have an alteredglycosylation pattern, wherein the N-linked glycans attached theretohave a reduction in the attachment of plant-specific xylose andplant-specific fucose residues and which comprise the terminal galactoseresidues (i.e., G2 glycan species). In this manner, glycoproteins thathave a substantially homogeneous profile for the G2 glycan species,and/or which are substantially homogenous for the G2 glycoform can beobtained from transgenic plants of the invention.

In other embodiments, it may be desirable to further modify theglycosylation pattern of the glycoproteins of the invention, wherein theN-linked glycans attached thereto further comprise a terminal sialicacid residue attached to one or both of the galactose residues attachedto the 1,3 and 1,6 mannose arms. The addition of the terminal sialicacid residue(s) may be required for the sustained stability, and in somecases function, of some therapeutic proteins.

Depending upon the transgenic plant system, natural sialylation ofglycoproteins may occur. Thus, there have been reports in the literaturethat cultured Arabidopsis, tobacco, and Medicago cultured cellssynthesize sialylated glycoproteins (Shah et al. (2003) Nat. Biotech.21(12):1470-1471; Joshi and Lopez (2005) Curr. Opin. Plant Biol.8(2):223-226). More recently, it was reported that Japanese rice expressactive sialyltransferase-like proteins (Takashima et al. (2006) J.Biochem. (Tokyo) 139(2):279-287). Hence, there are now orthogonalreports that plants have the machinery required to sialylateglycoproteins.

Where further modification of the glycosylation pattern of theglycoproteins of the invention, wherein the N-linked glycans attachedthereto further comprise a terminal sialic acid residue attached to oneor both of the galactose residues attached to the 1,3 and 1,6 mannosearms, is desired, the transgenic plants of the invention can be modifiedto express a β-1,4 galactosyltransferase, for example, human β-1,4galactosyltransferase, and to express or overexpress asialyltransferase. Thus, for example, the transgenic plants can befurther modified to express a sialyltransferase such as α-2,3- and/orα-2,6-sialyltransferase. See, for example WO 2004/071177; and Wee et al.(1998) Plant Cell 10:1759-1768; herein incorporated by reference intheir entirety. Alternatively, the transgenic plants of the inventioncan be modified to express a β-1,4 galactosyltransferase, for example,human β-1,4 galactosyltransferase, and to express any other enzymes thatare deficient in the plant host's sialic acid pathway. The strategy(s)employed can be determined after an initial investigation of whether theparticular plant host, for example, a duckweed, naturally expressessialic acid-containing N-glycans on native or recombinantly producedglycoproteins. For example, if there is not evidence for the presence ofthe terminal sialic acid residues on N-glycans of glycoproteins producedwithin the transgenic plant host, particularly a transgenic plant hostengineered to express a β-1,4 galactosyltransferase, then one or both ofthese strategies could be employed to achieve terminal sialylation ofthe N-glycans of glycoproteins produced within the transgenic plant hostof interest.

Alternatively, the glycoprotein compositions of the invention that aresubstantially homologous for G2 glycan species or the G2 glycoform canbe modified by in vitro enzymatic processing; see, for example, U.S.Patent Application Publication No. 20030040037; herein incorporated byreference in its entirety.

It is also recognized that for some glycoproteins produced in thetransgenic plants of the invention, it may be desirable to have themammalian α1-6 fucose residue attached to the trimannose core structure(Man₃GlcNAc₂) of the N-glycan species attached thereto. In suchembodiments, the transgenic plants of the invention can be furthergenetically modified to express an α1-6 fucosyltransferase, for example,human α1-6 fucosyltransferase, using glycoengineering methods known inthe art.

It is recognized that the glycoprotein compositions of the invention canbe produced by engineering any host cell of interest, including theplant host cells exemplified and described herein. In this manner, otherprotein expression host systems in addition to plant hosts, includinganimal, insect, bacterial cells and the like may be used to produceglycoprotein compositions according to the present invention. Suchprotein expression host systems may be engineered or selected to expressa predominant glycoform or alternatively may naturally produceglycoproteins having predominant glycan structures. Examples ofengineered protein expression host systems producing a glycoproteinhaving a predominant glycoform include gene knockouts/mutations (Shieldset al. (2002) JBC 277:26733-26740); genetic engineering (Umana et al.(1999) Nature Biotech. 17:176-180); or a combination of both.Alternatively, certain cells naturally express a predominant glycoform,for example, chickens, humans, and cows (Raju et al. (2000) Glycobiology10:477-486). Thus, the expression of a glycoprotein, including animmunoglobulin such as a monoclonal antibody, or composition havingpredominantly one specific glycan structure according to the presentinvention can be obtained by one skilled in the art by selecting atleast one of many expression host systems. Further expression hostsystems found in the art for production of glycoproteins include: CHOcells (see, for example, WO 9922764A1 and WO 03/035835A1); hybridomacells (Trebak et al. (1999) J. Immunol. Methods 230:59-70); insect cells(Hsu et al. (1997) JBC 272:9062-970). See also, WO 04/074499A2 regardingadditional plant host systems.

The glycoproteins produced in accordance with the methods of the presentinvention can be harvested from host cells in which they arerecombinantly produced in order to obtain them in their isolated orpurified form. In this manner, the recombinantly produced glycoproteinsof the invention are isolated from the host cells using any conventionalmeans known in the art and purified, for example, by chromatography,electrophoresis, dialysis, solvent-solvent extraction, and the like.Thus, the present invention also provides for purified glycoproteins,including monoclonal antibody compositions, where the glycoproteins havesubstantially homogeneous glycosylation profiles, and are substantiallyhomogeneous for the G0 glycoform. These purified glycoproteins aresubstantially free of host cellular material, and include preparationsof glycoprotein having less than about 30%, 20%, 10%, 5%, or 1% (by dryweight) of contaminating protein, as noted herein above. In someembodiments, these purified glycoproteins can include at least 0.001%,0.005%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%,6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up toabout 30% (by dry weight) of contaminating protein. Furthermore, for therecombinantly produced purified glycoproteins of the invention,optimally culture medium represents less than about 30%, 20%, 10%, 5%,or 1% (by dry weight) of chemical precursors or non-protein-of-interestchemicals within the purified glycoprotein preparation, as noted hereinabove. Thus, in some embodiments, culture medium components within thesepurified glycoproteins can represent at least 0.001%, 0.005%, 0.1%,0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%,7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or up to about 30% (by dryweight) of chemical precursors or non-protein-of-interest chemicalswithin the purified glycoprotein preparation. In some embodiments,isolation and purification results in recovery of purified glycoproteinthat is free of contaminating host protein, free of culture mediumcomponents, and/or free of both contaminating host protein and culturemedium components.

Thus, in some embodiments, the protein expression host system is aplant, for example, a duckweed, and the purified glycoprotein obtainedfrom the plant host is substantially free of plant cellular material,including embodiments where the preparations of glycoprotein have lessthan about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminatingplant protein. In other embodiments, the plant culture medium representsless than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals within the purifiedglycoprotein.

In some embodiments, these purified glycoproteins obtained from theplant host can include at least 0.001%, 0.005%, 0.1%, 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%,9%, 9.5%, 10%, 15%, 20%, 25%, or up to about 30% (by dry weight) ofcontaminating plant protein. In other embodiments, plant culture mediumcomponents within in these purified glycoproteins can represent at least0.001%, 0.005%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%,5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or upto about 30% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals within the purified glycoprotein. Insome embodiments, isolation and purification from the plant host resultsin recovery of purified glycoprotein that is free of contaminating plantprotein, free of plant culture medium components, and/or free of bothcontaminating plant protein and plant culture medium components.

Expression Cassettes

According to the present invention, stably transformed higher plants,for example, stably transformed duckweed, are obtained by transformationwith a polynucleotide of interest contained within an expressioncassette. Depending upon the objective, the polynucleotide of interestcan be one encoding a FucT or XylT polypeptide of interest, for example,encoding the polypeptide set forth in SEQ ID NO:3 (FucT) or SEQ ID NO:6or 21 (XylT), or a variant thereof, thus providing for expression ofthese polypeptides in a cell, for example, a plant cell, or can be aFucT or XylT inhibitory polynucleotide that is capable of inhibitingexpression or function of the FucT or XylT polypeptide when stablyintroduced into a cell, for example, a plant cell of interest.

Thus, in some embodiments, the FucT and/or XylT polynucleotides of theinvention, including those set forth in SEQ ID NOS:1 and 2 (FucT) andSEQ ID NOS:4, 5, 19, and 20 (XylT) and fragments and variants thereof,are used to construct expression cassettes that comprise a FucT and/orXylT inhibitory polynucleotide as defined herein above. Stablyintroducing such an expression cassette into a plant or plant cell ofinterest can provide for inhibition of expression or function of theFucT and/or XylT polypeptides of the invention, including those setforth in SEQ ID NO:3 (FucT) and SEQ ID NO:6 or 21 (XylT) and variantsthereof, thereby altering the N-glycan glycosylation pattern ofendogenous and heterologous glycoproteins within a plant or plant cellstably transformed with the expression cassette.

In some embodiments, the plant or plant cell that is stably transformedwith an expression cassette comprising a FucT and/or XylT inhibitorypolynucleotide has also been stably transformed with an expressioncassette that provides for expression of a heterologous polypeptide ofinterest, for example, a mammalian protein of interest, including themammalian proteins of pharmaceutical interest as noted herein above. Theexpression cassette providing for expression of a heterologouspolypeptide of interest can be provided on the same polynucleotide (forexample, on the same transformation vector) for introduction into aplant, or on a different polynucleotide (for example, on differenttransformation vectors) for introduction into the plant or plant cell ofinterest at the same time or at different times, by the same or bydifferent methods of introduction, for example, by the same or differenttransformation methods.

The expression cassettes of the present invention comprise expressioncontrol elements that at least comprise a transcriptional initiationregion (e.g., a promoter) linked to the polynucleotide of interest,i.e., a polynucleotide encoding a FucT or XylT polypeptide of theinvention, a FucT and/or XylT inhibitory polynucleotide, or apolynucleotide encoding a heterologous polypeptide of interest, forexample, a mammalian protein. Such an expression cassette is providedwith a plurality of restriction sites for insertion of thepolynucleotide or polynucleotides of interest (e.g., one polynucleotideof interest, two polynucleotides of interest, etc.) to be under thetranscriptional regulation of the promoter and other expression controlelements. In particular embodiments of the invention, the polynucleotideto be transferred contains two or more expression cassettes, each ofwhich encodes at least one polynucleotide of interest.

By “expression control element” is intended a regulatory region of DNA,usually comprising a TATA box, capable of directing RNA polymerase II,or in some embodiments, RNA polymerase III, to initiate RNA synthesis atthe appropriate transcription initiation site for a particular codingsequence. An expression control element may additionally comprise otherrecognition sequences generally positioned upstream or 5′ to the TATAbox, which influence (e.g., enhance) the transcription initiation rate.Furthermore, an expression control element may additionally comprisesequences generally positioned downstream or 3′ to the TATA box, whichinfluence (e.g., enhance) the transcription initiation rate.

The transcriptional initiation region (e.g., a promoter) may be nativeor homologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign, it is intendedthat the transcriptional initiation region is not found in the wild-typehost into which the transcriptional initiation region is introduced. By“functional promoter” is intended the promoter, when operably linked toa sequence encoding a protein of interest, is capable of drivingexpression (i.e., transcription and translation) of the encoded protein,or, when operably linked to an inhibitory sequence encoding aninhibitory nucleotide molecule (for example, a hairpin RNA,double-stranded RNA, miRNA polynucleotide, and the like), the promoteris capable of initiating transcription of the operably linked inhibitorysequence such that the inhibitory nucleotide molecule is expressed. Thepromoters can be selected based on the desired outcome. Thus theexpression cassettes of the invention can comprise constitutive,tissue-preferred, or other promoters for expression in plants.

As used herein a chimeric gene comprises a coding sequence operablylinked to a transcription initiation region that is heterologous to thecoding sequence.

Any suitable promoter known in the art can be employed according to thepresent invention, including bacterial, yeast, fungal, insect,mammalian, and plant promoters. For example, plant promoters, includingduckweed promoters, may be used. Exemplary promoters include, but arenot limited to, the Cauliflower Mosaic Virus 35S promoter, the opinesynthetase promoters (e.g., nos, mas, ocs, etc.), the ubiquitinpromoter, the actin promoter, the ribulose bisphosphate (RubP)carboxylase small subunit promoter, and the alcohol dehydrogenasepromoter. The duckweed RubP carboxylase small subunit promoter is knownin the art (Silverthorne et al. (1990) Plant Mol. Biol. 15:49). Otherpromoters from viruses that infect plants, preferably duckweed, are alsosuitable including, but not limited to, promoters isolated from Dasheenmosaic virus, Chlorella virus (e.g., the Chlorella virus adeninemethyltransferase promoter; Mitra et al. (1994) Plant Mol. Biol. 26:85),tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus,tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus,peanut stump virus, alfalfa mosaic virus, sugarcane baciliformbadnavirus and the like.

Other suitable expression control elements are disclosed in the commonlyowned and copending provisional application entitled “Expression ControlElements from the Lemnaceae Family,” assigned U.S. Patent ApplicationNo. 60/759,308, Attorney Docket No. 040989/243656, filed Jan. 17, 2006,herein incorporated by reference in its entirety. The expression controlelements disclosed in this copending application were isolated fromubiquitin genes for several members of the Lemnaceae family, and arethus referred to as “Lemnaceae ubiquitin expression control elements.”SEQ ID NO:7 of the present application sets forth the full-length Lemnaminor ubiquitin expression control element, including both the promoterplus 5′ UTR (nucleotides 1-1625) and intron (nucleotides 1626-2160). SEQID NO:8 sets forth the full-length Spirodella polyrrhiza ubiquitinexpression control element, including both the promoter plus 5′ UTR(nucleotides 1-1041) and intron (nucleotides 1042-2021). SEQ ID NO:9sets forth the full-length Lemna aequinoctialis ubiquitin expressioncontrol element, including both the promoter plus 5′ UTR (nucleotides1-964) and intron (nucleotides 965-2068). SEQ ID NO:10 sets forth thepromoter plus 5′ UTR portion of the L. minor ubiquitin expressioncontrol element (designated “LmUbq promoter” herein). SEQ ID NO:11 setsforth the promoter plus 5′ UTR portion of the S. polyrrhiza ubiquitinexpression control element (designated “SpUbq promoter” herein). SEQ IDNO:12 sets forth the promoter plus 5′ UTR portion of the L.aequinoctialis ubiquitin expression control element (designated “LaUbqpromoter” herein). SEQ ID NO:13 sets forth the intron portion of the L.minor ubiquitin expression control element (designated “LmUbq intron”herein). SEQ ID NO:14 sets forth the intron portion of the S. polyrrhizaubiquitin expression control element (designated “SpUbq intron” herein).SEQ ID NO:15 sets forth the intron portion of the L. aequinoctialisubiquitin expression control element (designated “LaUbq intron” herein).It is recognized that the individual promoter plus 5′ UTR sequences setforth in SEQ ID NOs:10-12, and biologically active variants andfragments thereof, can be used to regulate transcription of operablylinked nucleotide sequences of interest in plants. Similarly, one ormore of the intron sequences set forth in SEQ ID NOs:13-15, andbiologically active fragments or variants thereof, can be operablylinked to a promoter of interest, including a promoter set forth in SEQID NO:10, 11, or 12 in order to enhance expression of a nucleotidesequence that is operably linked to that promoter.

Fragments and variants of the disclosed expression control elements canalso be used within expression cassettes to drive expression of theoperably linked polynucleotide of interest. By “fragment of anexpression control element” is intended a portion of the full-lengthexpression control element, such as a portion of any one of theexpression control elements set forth in SEQ ID NOs:7-9. Fragments of anexpression control element retain biological activity and henceencompass fragments capable of initiating or enhancing expression of anoperably linked polynucleotide of interest. Thus, for example, less thanthe entire expression control elements disclosed herein may be utilizedto drive expression of an operably linked polynucleotide of interest.Specific, non-limiting examples of such fragments of an expressioncontrol element include the nucleotide sequences set forth in any one ofSEQ ID NOs:10-12 (as described herein above), as well as 5′ truncationsof the L. minor ubiquitin expression control element (SEQ ID NO:7), suchas nucleotides 1288-2160 of SEQ ID NO:7 (LmUbq truncated promoter No. 1)and nucleotides 1132-2160 of SEQ ID NO:1 (LmUbq truncated promoter No.2). See the copending provisional application assigned U.S. PatentApplication No. 60/759,308, herein incorporated by reference in itsentirety.

The nucleotides of such fragments will usually comprise the TATArecognition sequence of the particular expression control element. Suchfragments can be obtained by use of restriction enzymes to cleave thenaturally occurring expression control elements disclosed herein; bysynthesizing a nucleotide sequence from the naturally occurring sequenceof the expression control element DNA sequence; or can be obtainedthrough the use of polymerase chain reaction (PCR) technology. Seeparticularly, Mullis et al. (1987) Methods Enzymol. 155:335-350, andErlich, ed. (1989) PCR Technology (Stockton Press, New York).

Variants of expression control elements, such as those resulting fromsite-directed mutagenesis, can also be used in the expression cassettesof the present invention to provide expression of the operably linkedpolynucleotide of interest. By “variant of an expression controlelement” is intended sequences having substantial similarity with anexpression control element disclosed herein (for example, the expressioncontrol element set forth in SEQ ID NO:7, 9, or 9), or with a fragmentthereof (for example, the respective sequences set forth in SEQ IDNOs:10-15). Naturally occurring variants of expression control elementscan be identified with the use of well-known molecular biologytechniques, as, for example, with PCR and hybridization techniques asoutlined above. Variant expression control elements also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis. Generally, variants of aparticular expression control element disclosed herein, includingvariants of SEQ ID NOs:7-15, will have at least 40%, 50%, 60%, 65%, 70%,generally at least 75%, 80%, 85%, preferably about 90%, 91%, 92%, 93%,94%, to 95%, 96%, 97%, and more preferably about 98%, 99% or moresequence identity to that particular nucleotide sequence as determinedby sequence alignment programs described herein above using defaultparameters.

Expression control elements, including promoters, can be chosen to givea desired level of regulation. For example, in some instances, it may beadvantageous to use a promoter that confers constitutive expression(e.g, the mannopine synthase promoter from Agrobacterium tumefaciens).Alternatively, in other situations, for example, where expression of aheterologous polypeptide is concerned, it may be advantageous to usepromoters that are activated in response to specific environmentalstimuli (e.g., heat shock gene promoters, drought-inducible genepromoters, pathogen-inducible gene promoters, wound-inducible genepromoters, and light/dark-inducible gene promoters) or plant growthregulators (e.g., promoters from genes induced by abscissic acid,auxins, cytokinins, and gibberellic acid). As a further alternative,promoters can be chosen that give tissue-specific expression (e.g.,root, leaf, and floral-specific promoters).

The overall strength of a given promoter can be influenced by thecombination and spatial organization of cis-acting nucleotide sequencessuch as upstream activating sequences. For example, activatingnucleotide sequences derived from the Agrobacterium tumefaciens octopinesynthase gene can enhance transcription from the Agrobacteriumtumefaciens mannopine synthase promoter (see U.S. Pat. No. 5,955,646 toGelvin et al.). In the present invention, the expression cassette cancontain activating nucleotide sequences inserted upstream of thepromoter sequence to enhance the expression of the nucleotide sequenceof interest. In one embodiment, the expression cassette includes threeupstream activating sequences derived from the Agrobacterium tumefaciensoctopine synthase gene operably linked to a promoter derived from anAgrobacterium tumefaciens mannopine synthase gene (see U.S. Pat. No.5,955,646, herein incorporated by reference).

Where the expression control element will be used to drive expression ofan operably linked DNA sequence encoding a small hpRNA molecule, forexample, within an RNAi expression cassette described herein above, itis advantageous to use an expression control element comprising apromoter recognized by the DNA dependent RNA polymerase III. As usedherein, “a promoter recognized by the DNA dependent RNA polymerase III”is a promoter which directs transcription of the associated DNA regionthrough the polymerase action of RNA polymerase III. These include genesencoding 5S RNA, tRNA, 7SL RNA, U6 snRNA and a few other small stableRNAs, many involved in RNA processing. Most of the promoters used by PolIII require sequence elements downstream of +1, within the transcribedregion. A minority of pol III templates however, lack any requirementfor intragenic promoter elements. These are referred to as type 3promoters. By “type 3 Pol III promoters” is intended those promotersthat are recognized by RNA polymerase III and contain all cis-actingelements, interacting with the RNA polymerase III upstream of the regionnormally transcribed by RNA polymerase III. Such type 3 Pol IIIpromoters can be assembled within the RNAi expression cassettes of theinvention to drive expression of the operably linked DNA sequenceencoding the small hpRNA molecule.

Typically, type 3 Pol III promoters contain a TATA box (located between−25 and −30 in Human U6 snRNA gene) and a Proximal Sequence element(PSE; located between −47 and −66 in Human U6 snRNA). They may alsocontain a Distal Sequence Element (DSE; located between −214 and −244 inHuman U6 snRNA). Type 3 Pol III promoters can be found, e.g., associatedwith the genes encoding 7SL RNA, U3 snRNA and U6 snRNA. Such sequenceshave been isolated from Arabidopsis, rice, and tomato. See, for example,SEQ ID NOs:1-8 of U.S. Patent Application Publication No. 20040231016.

Other nucleotide sequences for type 3 Pol III promoters can be found innucleotide sequence databases under the entries for the A. thaliana geneAT7SL-1 for 7SL RNA (X72228), A. thaliana gene AT7SL-2 for 7SL RNA(X72229), A. thaliana gene AT7SL-3 for 7SL RNA (AJ290403), Humuluslupulus H17SL-1 gene (AJ236706), Humulus lupulus H17SL-2 gene(AJ236704), Humulus lupulus H17SL-3 gene (AJ236705), Humulus lupulusH17SL-4 gene (AJ236703), A. thaliana U6-1 snRNA gene (X52527), A.thaliana U6-26 snRNA gene (X52528), A. thaliana U6-29 snRNA gene(X52529), A. thaliana U6-1 snRNA gene (X52527), Zea mays U3 snRNA gene(Z29641), Solanum tuberosum U6 snRNA gene (Z17301; X60506; S83742),tomato U6 smal nuclear RNA gene (X51447), A. thaliana U3C snRNA gene(X52630), A. thaliana U3B snRNA gene (X52629), Oryza sativa U3 snRNApromoter (X79685), tomato U3 small nuclear RNA gene (X14411), Triticumaestivum U3 snRNA gene (X63065), and Triticum aestivum U6 snRNA gene(X63066).

Other type 3 Pol III promoters may be isolated from other varieties oftomato, rice or Arabidopsis, or from other plant species using methodswell known in the art. For example, libraries of genomic clones fromsuch plants may be isolated using U6 snRNA, U3 snRNA, or 7SL RNA codingsequences (such as the coding sequences of any of the above mentionedsequences identified by their accession number and additionally theVicia faba U6snRNA coding sequence (X04788), the maize DNA for U6 snRNA(X52315), or the maize DNA for 7SL RNA (X14661)) as a probe, and theupstream sequences, preferably the about 300 to 400 bp upstream of thetranscribed regions may be isolated and used as type 3 Pol IIIpromoters. Alternatively, PCR based techniques such as inverse-PCR orTAIL™-PCR may be used to isolate the genomic sequences including thepromoter sequences adjacent to known transcribed regions. Moreover, anyof the type 3 Pol III promoter sequences described herein, identified bytheir accession numbers and SEQ ID NOS, may be used as probes understringent hybridization conditions or as source of information togenerate PCR primers to isolate the corresponding promoter sequencesfrom other varieties or plant species.

Although type 3 Pol III promoters have no requirement for cis-actingelements located with the transcribed region, it is clear that sequencesnormally located downstream of the transcription initiation site maynevertheless be included in the RNAi expression cassettes of theinvention. Further, while type 3 Pol III promoters originally isolatedfrom monocotyledonous plants can effectively be used in RNAi expressioncassettes to suppress expression of a target gene in both dicotyledonousand monocotyledonous plant cells and plants, type 3 Pol III promotersoriginally isolated from dicotyledonous plants reportedly can only beefficiently used in dicotyledonous plant cells and plants. Moreover, themost efficient gene silencing reportedly is obtained when the RNAiexpression cassette is designed to comprise a type 3 Pol III promoterderived from the same or closely related species. See, for example, U.S.Patent Application Publication No. 20040231016. Thus, where the plant ofinterest is a monocotyledonous plant, and small hpRNA interference isthe method of choice for inhibiting expression of FucT and/or XylT, thetype 3 Pol III promoter preferably is from another monocotyledonousplant, including the plant species for which the glycosylation patternof N-linked glycans of a glycoprotein of interest is to be altered.

The expression cassette of the invention thus includes in the 5′-3′direction of transcription, an expression control element comprising atranscriptional and translational initiation region, a polynucleotide ofinterest, for example, a sequence encoding a heterologous protein ofinterest or a sequence encoding a FucT or XylT inhibitory sequence that,when expressed, is capable of inhibiting the expression or function ofFucT and/or XylT, and a transcriptional and translational terminationregion functional in plants. Any suitable termination sequence known inthe art may be used in accordance with the present invention. Thetermination region may be native with the transcriptional initiationregion, may be native with the nucleotide sequence of interest, or maybe derived from another source. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthetase and nopaline synthetase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262:141; Proudfoot (1991) Cell64:671; Sanfacon et al. (1991) Genes Dev. 5:141; Mogen et al. (1990)Plant Cell 2:1261; Munroe et al. (1990) Gene 91:151; Ballas et al.(1989) Nucleic Acids Res. 17:7891; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627. Additional exemplary termination sequences are the peaRubP carboxylase small subunit termination sequence and the CauliflowerMosaic Virus 35S termination sequence. Other suitable terminationsequences will be apparent to those skilled in the art, including theoligo dT stretch disclosed herein above for use with type 3 Pol IIIpromoters driving expression of a FucT and/or XylT inhibitorypolynucleotide that forms a small hpRNA structure.

Alternatively, the polynucleotide(s) of interest can be provided on anyother suitable expression cassette known in the art.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells or tissues. Selectablemarker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds. Herbicide resistance genes generally code for amodified target protein insensitive to the herbicide or for an enzymethat degrades or detoxifies the herbicide in the plant before it canact. See DeBlock et al. (1987) EMBO J. 6:2513; DeBlock et al. (1989)Plant Physiol. 91:691; Fromm et al. (1990) BioTechnology 8:833;Gordon-Kamm et al. (1990) Plant Cell 2:603. For example, resistance toglyphosphate or sulfonylurea herbicides has been obtained using genescoding for the mutant target enzymes, 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) and acetolactate synthase (ALS). Resistance toglufosinate ammonium, boromoxynil, and 2,4-dichlorophenoxyacetate(2,4-D) have been obtained by using bacterial genes encodingphosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides.

For purposes of the present invention, selectable marker genes include,but are not limited to, genes encoding neomycin phosphotransferase II(Fraley et al. (1986) CRC Critical Reviews in Plant Science 4:1);cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci.USA 88:4250); aspartate kinase; dihydrodipicolinate synthase (Perl etal. (1993) BioTechnology 11:715); bar gene (Toki et al. (1992) PlantPhysiol. 100:1503; Meagher et al. (1996) Crop Sci. 36:1367); tryptophandecarboxylase (Goddijn et al. (1993) Plant Mol. Biol. 22:907); neomycinphosphotransferase (NEO; Southern et al. (1982) J. Mol. Appl. Gen.1:327); hygromycin phosphotransferase (HPT or HYG; Shimizu et al. (1986)Mol. Cell. Biol. 6:1074); dihydrofolate reductase (DHFR; Kwok et al.(1986) Proc. Natl. Acad. Sci. USA 83:4552); phosphinothricinacetyltransferase (DeBlock et al. (1987) EMBO J. 6:2513);2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al.(1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (U.S. Pat.No. 4,761,373 to Anderson et al.; Haughn et al. (1988) Mol. Gen. Genet.221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al.(1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalker etal.); acetyl-coenzyme A carboxylase (Parker et al. (1990) Plant Physiol.92:1220); dihydropteroate synthase (sulI; Guerineau et al. (1990) PlantMol. Biol. 15:127); and 32 kDa photosystem II polypeptide (psbA;Hirschberg et al. (1983) Science 222:1346 (1983).

Also included are genes encoding resistance to: gentamycin (e.g., aacC1,Wohlleben et al. (1989) Mol. Gen. Genet. 217:202-208); chloramphenicol(Herrera-Estrella et al. (1983) EMBO J. 2:987); methotrexate(Herrera-Estrella et al. (1983) Nature 303:209; Meijer et al. (1991)Plant Mol. Biol. 16:807); hygromycin (Waldron et al. (1985) Plant Mol.Biol. 5:103; Zhijian et al. (1995) Plant Science 108:219; Meijer et al.(1991) Plant Mol. Bio. 16:807); streptomycin (Jones et al. (1987) Mol.Gen. Genet. 210:86); spectinomycin (Bretagne-Sagnard et al. (1996)Transgenic Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol.7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127);bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D (Streber etal. (1989) BioTechnology 7:811); phosphinothricin (DeBlock et al. (1987)EMBO J. 6:2513); spectinomycin (Bretagne-Sagnard and Chupeau, TransgenicResearch 5:131).

The bar gene confers herbicide resistance to glufosinate-typeherbicides, such as phosphinothricin (PPT) or bialaphos, and the like.As noted above, other selectable markers that could be used in thevector constructs include, but are not limited to, the pat gene, alsofor bialaphos and phosphinothricin resistance, the ALS gene forimidazolinone resistance, the HPH or HYG gene for hygromycin resistance,the EPSP synthase gene for glyphosate resistance, the Hm1 gene forresistance to the Hc-toxin, and other selective agents used routinelyand known to one of ordinary skill in the art. See Yarranton (1992)Curr. Opin. Biotech. 3:506; Chistopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314; Yao et al. (1992) Cell 71:63; Reznikoff (1992)Mol. Microbiol. 6:2419; Barkley et al. (1980) The Operon 177-220; Hu etal. (1987) Cell 48:555; Brown et al. (1987) Cell 49:603; Figge et al.(1988) Cell 52:713; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA86:5400; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549;Deuschle et al. (1990) Science 248:480; Labow et al. (1990) Mol. Cell.Biol. 10:3343; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072; Wyborskiet al. (1991) Nuc. Acids Res. 19:4647; Hillenand-Wissman (1989) Topicsin Mol. And Struc. Biol. 10:143; Degenkolb et al. (1991) Antimicrob.Agents Chemother. 35:1591; Kleinschnidt et al. (1988) Biochemistry27:1094; Gatz et al. (1992) Plant J. 2:397; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA 89:5547; Oliva et al. (1992) Antimicrob. AgentsChemother. 36:913; Hlavka et al. (1985) Handbook of ExperimentalPharmacology 78; and Gill et al. (1988) Nature 334:721. Such disclosuresare herein incorporated by reference.

The above list of selectable marker genes are not meant to be limiting.Any selectable marker gene can be used in the present invention.

Modification of Nucleotide Sequences for Enhanced Expression in a PlantHost

Where the plant of interest is also genetically modified to express aheterologous protein of interest, for example, a transgenic plant hostserving as an expression system for recombinant production of aheterologous protein, the present invention provides for themodification of the expressed polynucleotide sequence encoding theheterologous protein of interest to enhance its expression in the hostplant. Thus, where appropriate, the polynucleotides may be optimized forincreased expression in the transformed plant. That is, thepolynucleotides can be synthesized using plant-preferred codons forimproved expression. See, for example, Campbell and Gowri (1990) PlantPhysiol. 92:1-11 for a discussion of host-preferred codon usage. Methodsare available in the art for synthesizing nucleotide sequences withplant-preferred codons. See, e.g., U.S. Pat. Nos. 5,380,831 and5,436,391; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 15:3324;Iannacome et al. (1997) Plant Mol. Biol. 34:485; and Murray et al.,(1989) Nucleic Acids. Res. 17:477, herein incorporated by reference.

In some embodiments of the invention, the plant host is a member of theduckweed family, and the polynucleotide encoding the heterologouspolypeptide of interest, for example, a mammalian polypeptide, ismodified for enhanced expression of the encoded heterologouspolypeptide. In this manner, one such modification is the synthesis ofthe polynucleotide encoding the heterologous polypeptide of interestusing duckweed-preferred codons, where synthesis can be accomplishedusing any method known to one of skill in the art. The preferred codonsmay be determined from the codons of highest frequency in the proteinsexpressed in duckweed. For example, the frequency of codon usage forLemna gibba is found on the web page:http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Lemna+gibba+[gbpln],and the frequency of codon usage for Lemna minor is found on the webpagehttp://www.kazusa.or.jp/codon/cgibin/showcodon.cgi?species=Lemna+minor+[gbpln]and in Table 1. It is recognized that heterologous genes that have beenoptimized for expression in duckweed and other monocots, as well asother dicots, can be used in the methods of the invention. See, e.g., EP0 359 472, EP 0 385 962, WO 91/16432; Perlak et al. (1991) Proc. Natl.Acad. Sci. USA 88:3324; Iannacome et al. (1997) Plant Mol. Biol. 34:485;and Murray et al. (1989) Nuc. Acids Res. 17:477, and the like, hereinincorporated by reference. It is further recognized that all or any partof the polynucleotide encoding the heterologous polypeptide of interestmay be optimized or synthetic. In other words, fully optimized orpartially optimized sequences may also be used. For example, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% of the codons may be duckweed-preferredcodons. In one embodiment, between 90 and 96% of the codons areduckweed-preferred codons. The coding sequence of a polynucleotidesequence encoding a heterologous polypeptide of interest may comprisecodons used with a frequency of at least 17% in Lemna gibba. In oneembodiment, the modified nucleotide sequence is the humanα-2B-interferon encoding nucleotide sequence shown in SEQ ID NO:16,which contains 93% duckweed preferred codons.

TABLE 1 Lemna gibba-preferred codons from GenBank Release 113 UUU 2.2(4) UCU 0.5 (1) UAU 2.2 (4) UGU 0.0 (0) UUC 50.5 (92) UCC 31.9 (58) UAC40.1 (73) UGC 17.6 (32) UUA 0.0 (0) UCA 0.5 (1) UAA 3.8 (7) UGA 1.6 (3)UUG 2.7 (5) UCG 15.4 (28) UAG 0.0 (0) UGG 24.2 (44) CUU 0.5 (1) CCU 6.6(12) CAU 0.5 (1) CGU 1.1 (2) CUC 39.0 (71) CCC 43.4 (79) CAC 6.6 (12)CGC 26.9 (49) CUA 1.1 (2) CCA 2.2 (4) CAA 4.4 (8) CGA 1.1 (2) CUG 22.5(41) CCG 20.9 (38) CAG 26.9 (49) CGG 7.7 (14) AUU 0.0 (0) ACU 3.3 (6)AAU 1.1 (2) AGU 0.0 (0) AUC 33.5 (61) ACC 26.4 (48) AAC 37.9 (69) AGC22.0 (40) AUA 0.0 (0) ACA 0.5 (1) AAA 0.0 (0) AGA 4.9 (9) AUG 33.5 (61)ACG 9.3 (17) AAG 57.1 (104) AGG 6.0 (11) GUU 9.3 (17) GCU 7.1 (13) GAU1.6 (3) GGU 1.1 (2) GUC 28.0 (51) GCC 73.6 (134) GAC 38.4 (70) GGC 46.7(85) GUA 0.0 (0) GCA 5.5 (10) GAA 2.2 (4) GGA 1.1 (2) GUG 34.0 (62) GCG20.9 (38) GAG 62.6 (114) GGG 27.5 (50)

Other modifications can also be made to the polynucleotide encoding theheterologous polypeptide of interest to enhance its expression in aplant host of interest, including duckweed. These modifications include,but are not limited to, elimination of sequences encoding spuriouspolyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the polynucleotide encoding the heterologous polypeptide ofinterest may be modified to avoid predicted hairpin secondary mRNAstructures.

There are known differences between the optimal translation initiationcontext nucleotide sequences for translation initiation codons inanimals and plants and the composition of these translation initiationcontext nucleotide sequences can influence the efficiency of translationinitiation. See, for example, Lukaszewicz et al. (2000) Plant Science154:89-98; and Joshi et al. (1997); Plant Mol. Biol. 35:993-1001. In thepresent invention, the translation initiation context nucleotidesequence for the translation initiation codon of the polynucleotidenucleotide of interest, for example, the polynucleotide encoding aheterologous polypeptide of interest, may be modified to enhanceexpression in duckweed. In one embodiment, the nucleotide sequence ismodified such that the three nucleotides directly upstream of thetranslation initiation codon of the nucleotide sequence of interest are“ACC.” In a second embodiment, these nucleotides are “ACA.”

Expression of a transgene in a host plant, including duckweed, can alsobe enhanced by the use of 5′ leader sequences. Such leader sequences canact to enhance translation. Translation leaders are known in the art andinclude, but are not limited to, picornavirus leaders, e.g., EMCV leader(Encephalomyocarditis 5′ noncoding region; Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126); potyvirus leaders, e.g., TEV leader(Tobacco Etch Virus; Allison et al. (1986) Virology 154:9); humanimmunoglobulin heavy-chain binding protein (BiP; Macajak and Sarnow(1991) Nature 353:90); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke (1987) Nature325:622); tobacco mosaic virus leader (TMV; Gallie (1989) MolecularBiology of RNA, 23:56); potato etch virus leader (Tomashevskaya et al.(1993) J. Gen. Virol. 74:2717-2724); Fed-1 5′ untranslated region(Dickey (1992) EMBO J. 11:2311-2317); RbcS 5′ untranslated region(Silverthorne et al. (1990) J. Plant. Mol. Biol. 15:49-58); and maizechlorotic mottle virus leader (MCMV; Lommel et al. (1991) Virology81:382). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965.Leader sequence comprising plant intron sequence, including intronsequence from the maize alcohol dehydrogenase 1 (ADH1) gene, the castorbean catalase gene, or the Arabidopsis tryptophan pathway gene PAT1 hasalso been shown to increase translational efficiency in plants (Calliset al. (1987) Genes Dev. 1:1183-1200; Mascarenhas et al. (1990) PlantMol. Biol. 15:913-920). See also copending provisional application U.S.Patent Application No. 60/759,308, wherein leader sequence comprising aduckweed intron sequence selected from the group consisting of theintrons set forth in SEQ ID NOs:13-15 provides for increasedtranslational efficiency in duckweed.

In some embodiments of the present invention, nucleotide sequencecorresponding to nucleotides 1222-1775 of the maize alcoholdehydrogenase 1 gene (ADH1; GenBank Accession Number X04049), ornucleotide sequence corresponding to the intron set forth in SEQ IDNO:13, 14, or 15, is inserted upstream of the polynucleotide encodingthe heterologous polypeptide of interest or the FucT and/or XylTinhibitory polynucleotide to enhance the efficiency of its translation.In another embodiment, the expression cassette contains the leader fromthe Lemna gibba ribulose-bis-phosphate carboxylase small subunit 5B gene(RbcS leader; see Buzby et al. (1990) Plant Cell 2:805-814; also see SEQID NO:16, 17, or 18 of the present invention).

See also, by way of example only, the expression vectors disclosed inthe figures herein, wherein the RbcS leader and ADH1 intron are includedas upstream regulatory sequences within an expression cassettecomprising the FucT inhibitory polynucleotide (FIG. 8), the XylTinhibitory polynucleotide (FIGS. 9 and 11), an expression cassettecomprising the chimeric FucT/XylT inhibitory molecule (FIG. 10), or anexpression cassette comprising the coding sequence for the heterologouspolypeptide, the IgG1 heavy chain of a monoclonal antibody (FIGS. 12,13, and 14) or the light chain of a monoclonal antibody (FIG. 14);wherein the LmUbq promoter and LmUbq intron are included as upstreamregulatory sequences within an expression cassette comprising the FucTinhibitory polynucleotide (FIG. 11), or an expression cassettecomprising the coding sequence for the heterologous polypeptide, theIgF1 light chain of a monoclonal antibody (FIG. 13); wherein the SpUbqpromoter and SpUbq intron are included as upstream regulatory sequenceswithin an expression cassette comprising the FucT inhibitorypolynucleotide (FIG. 13), or an expression cassette comprising thechimeric FucT/XylT inhibitory polynucleotide (FIG. 12); and wherein theLaUbq promoter and LaUbq intron are included as upstream regulatorysequences in an expression cassette comprising the XylT inhibitorypolynucleotide (FIG. 13).

It is recognized that any of the expression-enhancing nucleotidesequence modifications described above can be used in the presentinvention, including any single modification or any possible combinationof modifications. The phrase “modified for enhanced expression” in aplant, for example, a duckweed plant, as used herein refers to apolynucleotide sequence that contains any one or any combination ofthese modifications.

Signal Peptides

It is recognized that the heterologous polypeptide of interest may beone that is normally or advantageously expressed as a secreted protein.Secreted proteins are usually translated from precursor polypeptidesthat include a “signal peptide” that interacts with a receptor proteinon the membrane of the endoplasmic reticulum (ER) to direct thetranslocation of the growing polypeptide chain across the membrane andinto the endoplasmic reticulum for secretion from the cell. This signalpeptide is often cleaved from the precursor polypeptide to produce a“mature” polypeptide lacking the signal peptide. In an embodiment of thepresent invention, a biologically active polypeptide is expressed in theplant host of interest, for example, duckweed or other higher plant,from a polynucleotide sequence that is operably linked with a nucleotidesequence encoding a signal peptide that directs secretion of thepolypeptide into the culture medium. Plant signal peptides that targetprotein translocation to the endoplasmic reticulum (for secretionoutside of the cell) are known in the art. See, for example, U.S. Pat.No. 6,020,169 to Lee et al. In the present invention, any plant signalpeptide can be used to target the expressed polypeptide to the ER.

In some embodiments, the signal peptide is the Arabidopsis thalianabasic endochitinase signal peptide (amino acids 14-34 of NCBI ProteinAccession No. BAA82823), the extensin signal peptide (Stiefel et al.(1990) Plant Cell 2:785-793), the rice α-amylase signal peptide (aminoacids 1-31 of NCBI Protein Accession No. AAA33885), or a modified riceα-amylase signal sequence (SEQ ID NO:17). In another embodiment, thesignal peptide corresponds to the signal peptide of a secreted duckweedprotein.

Alternatively, a mammalian signal peptide can be used to targetrecombinant polypeptides expressed in a genetically engineered plant ofthe invention, for example, duckweed or other higher plant of interest,for secretion. It has been demonstrated that plant cells recognizemammalian signal peptides that target the endoplasmic reticulum, andthat these signal peptides can direct the secretion of polypeptides notonly through the plasma membrane but also through the plant cell wall.See U.S. Pat. Nos. 5,202,422 and 5,639,947 to Hiatt et al. In oneembodiment of the present invention, the mammalian signal peptide thattargets polypeptide secretion is the human α-2b-interferon signalpeptide (amino acids 1-23 of NCBI Protein Accession No. AAB59402).

In one embodiment, the nucleotide sequence encoding the signal peptideis modified for enhanced expression in the plant host of interest, forexample, duckweed or other higher plant, utilizing any modification orcombination of modifications disclosed above for the polynucleotidesequence of interest.

The secreted biologically active polypeptide can be harvested from theculture medium by any conventional means known in the art and purifiedby chromatography, electrophoresis, dialysis, solvent-solventextraction, and the like. In this manner, purified polypeptides, asdefined above, can be obtained from the culture medium.

Thus, in some embodiments, the protein expression host system is aplant, for example, a duckweed or other higher plant, and the secretedbiologically active polypeptide is a glycoprotein of the invention,where the glycoprotein has a substantially homogeneous glycosylationprofile, and is substantially homogeneous for the G0 glycoform. In suchembodiments, any such glycoprotein that may remain within the plantmaterial can optionally be isolated and purified as described above. Thesecreted glycoprotein can be obtained from the plant culture medium andpurified using any conventional means in the art as noted above. In thismanner, the purified glycoprotein obtained from the plant material issubstantially free of plant cellular material, and includes embodimentswhere the preparations of glycoprotein have less than about 30%, 20%,10%, 5%, or 1% (by dry weight) of contaminating plant protein. Where thepurified glycoprotein is obtained from the plant culture medium, theplant culture medium represents less than about 30%, 20%, 10%, 5%, or 1%(by dry weight) of chemical precursors or non-protein-of-interestchemicals within the purified glycoprotein preparation.

In some embodiments, these purified glycoproteins obtained from theplant host can include at least 0.001%, 0.005%, 0.1%, 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%,9%, 9.5%, 10%, 15%, 20%, 25%, or up to about 30% (by dry weight) ofcontaminating plant protein. In other embodiments, where theglycoprotein is collected from the plant culture medium, the plantculture medium in these purified glycoproteins can include at least0.001%, 0.005%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%,5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, or upto about 30% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals within the purified glycoproteinpreparation. In some embodiments, isolation and purification from theplant host, and where secreted, from the culture medium, results inrecovery of purified glycoprotein that is free of contaminating plantprotein, free of plant culture medium components, and/or free of bothcontaminating plant protein and plant culture medium components.

Transformed Plants and Transformed Duckweed Plants and Duckweed NoduleCultures

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell or nodule, that is, monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plants orplant cells or nodules include microinjection (Crossway et al. (1986)Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055 and 5,981,840, both of whichare herein incorporated by reference), direct gene transfer (Paszkowskiet al. (1984) EMBO J. 3:2717-2722), ballistic particle acceleration(see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and5,932,782 (each of which is herein incorporated by reference); and Tomeset al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926). The cells that have beentransformed may be grown into plants in accordance with conventionalways. See, for example, McCormick et al. (1986) Plant Cell Reports5:81-84.

The stably transformed duckweed utilized in this invention can beobtained by any method known in the art. In one embodiment, the stablytransformed duckweed is obtained by one of the gene transfer methodsdisclosed in U.S. Pat. No. 6,040,498 to Stomp et al., hereinincorporated by reference. These methods include gene transfer byballistic bombardment with microprojectiles coated with a nucleic acidcomprising the nucleotide sequence of interest, gene transfer byelectroporation, and gene transfer mediated by Agrobacterium comprisinga vector comprising the nucleotide sequence of interest. In oneembodiment, the stably transformed duckweed is obtained via any one ofthe Agrobacterium-mediated methods disclosed in U.S. Pat. No. 6,040,498to Stomp et al. The Agrobacterium used is Agrobacterium tumefaciens orAgrobacterium rhizogenes.

It is preferred that the stably transformed duckweed plants utilized inthese methods exhibit normal morphology and are fertile by sexualreproduction. Preferably, transformed plants of the present inventioncontain a single copy of the transferred nucleic acid, and thetransferred nucleic acid has no notable rearrangements therein. Alsopreferred are duckweed plants in which the transferred nucleic acid ispresent in low copy numbers (i.e., no more than five copies,alternately, no more than three copies, as a further alternative, fewerthan three copies of the nucleic acid per transformed cell).

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Lemna, a small aquatic plant, is a scalable and economically attractiveexpression platform for the manufacture of therapeutic proteins free ofhuman pathogens and with a clear path towards regulatory approval. TheLemna expression system (LEX System^(SM)) enables rapid clonal expansionof transgenic plants, secretion of transgenic proteins, high proteinyields, ease of containment that is comparable to mammalian cell culturesystems such as CHO cells, and has the additional advantage of lowoperating and capital costs (Gasdaska et al. (2003) Bioprocessing J.50-56). In addition, this plant expression system offers the advantageof high protein yields (in the range of 6-8% of the total solubleprotein (TSP)). These expression levels, in combination with Lemna'shigh protein content and fast growth rate (36 hr doubling time), enableproduction of >1 g of mAb per kg biomass in a robust and well-controlledformat.

The following examples demonstrate how humanization of the glycosylationprofile of a mAb was accomplished by coexpression of the mAb with aninterference RNA (RNAi) construct targeting the endogenous expression ofα1,3-fucosyltransferase and β1,2-xylosyltransferase genes. The resultantmAb contained a single major N-glycan species (>95%) devoid of the plantspecific α-1,3-linked fucose and β-1,2-linked xylose sugars. In receptorbinding assays, this glycan optimized mAb exhibited enhanced effectorcell receptor binding activity when compared to mAb produced inwild-type Lemna having the native glycosylation machinery and mAbproduced in CHO cells.

Example 1 Isolation of Lemna minor Proteins Involved in N-Glycosylationof Proteins

In order to generate recombinant proteins with remodeled N-glycan, alpha1-3 fucosyltransferase and β1-2 xylosyltransferase were selected astargets for RNAi gene silencing in L. minor. Initial results from cDNAsequencing efforts indicated that two or more isoforms were present foreach of the target genes. Sequence homology between the isoforms wasdetermined to be between 90% and 95%. Full length cDNA sequences forboth target genes were retrieved and characterized. The full-length cDNAsequence, including 5′- and 3′-UTR, for L. minor α1-3 fucosyltransferase(FucT) is set forth in FIG. 1; see also SEQ ID NO:1 (open reading frameset forth in SEQ ID NO:2). The predicted amino acid sequence encodedthereby is set forth in SEQ ID NO:3. The encoded protein shares somesimilarity with other FucTs from other higher plants. See FIG. 2. Forexample, the L. minor FucT sequence shares approximately 50.1% sequenceidentity with the Arabidopsis thaliana FucT shown in FIG. 2.

The full-length cDNA sequence, including 5′- and 3′-UTR, for L. minorβ1-2 xylosyltransferase (XylT) (isoform #1) is set forth in FIG. 3; seealso SEQ ID NO:4 (ORF set forth in SEQ ID NO:5). The predicted aminoacid sequence encoded thereby is set forth in SEQ ID NO:6. The encodedprotein shares some similarity with other XylTs from other higherplants. See FIG. 4. For example, the L. minor XylT shares approximately56.4% sequence identity with the Arabidopsis thaliana XylT shown in FIG.4. A partial-length cDNA sequence, including 3′-UTR, for L. minor β1-2xylosyltransferase (XylT) (isoform #2) is set forth in FIG. 31; see alsoSEQ ID NO:19 (ORF set forth in SEQ ID NO:20). The predicted amino acidsequence encoded thereby is set forth in SEQ ID NO:21. Thepartial-length XylT isoform #2 shares high sequence identity with thecorresponding region of the full-length XylT isoform #1, as can be seenfrom the alignment shown in FIG. 32.

Example 2 RNAi Inhibition of Expression of L. minor FucT and XylT

Several RNAi strategies were undertaken to inhibit expression of the L.minor FucT and XylT isoforms. FIGS. 5-7, 33, and 34 outline thesestrategies. FIGS. 8-13 show maps of the various constructs that weremade to achieve the desired knockout of expression of these two genes. Anumber of transgenic lines comprising the various knockout RNAiconstructs were generated using standard transformation protocolsdescribed herein above.

The test antibody, designated herein as mAbI, was expressed in wild-typeLemna having the native glycosylation machinery, and transgenic Lemnalines expressing RNAi constructs designed to inhibit expression of L.minor XylT and FucT isoforms. Generally, three binary vectors wereconstructed for expression of mAbI in the Lemna system. Expressionvector mAbI01 contained codon optimized genes encoding heavy (H) andlight (L) chains of mAbI; vector mAbI04 contained codon optimized genesencoding mAbI H and L chains and a chimeric RNAi construct targetingexpression of both XylT and FucT isoforms; and vector mAbI05 containedcodon optimized genes encoding mAbI H and L chains, a single-gene RNAiconstruct targeting FucT gene expression, and a single-gene RNAiconstruct targeting XylT gene expression. Independent transgenic lineswere generated for the mABI01, mAbI04, and mAbI05 expression vectors.

Optimized genes for mAbI H and L chains were designed to have Lemnapreferred codon usage (63%-67% GC content) and contain the riceα-amylase signal sequence (GenBank M24286) fused to the 5′ end of theircoding sequences. Restriction endonuclease sites were added for cloninginto Agrobacterium binary vectors (EcoRI (5′)/SacI (3′), H-chain) and(SalI (5′)/HindIII (3′), L-chain).

For the XF02 data presented in FIGS. 15-17 and the mAbI04 data presentedin FIGS. 22-24 and 26, described herein below, the RNAi strategy forinhibiting expression of the L. minor FucT and XylT isoforms employedthe chimeric RNAi design shown in FIG. 34. For the mAbI05 data presentedin FIG. 27, described herein below, the RNAi strategy for inhibitingexpression of the L. minor FucT and XylT isoforms employed a doubleknockout of these genes using a combination of the single gene RNAidesigns shown in FIG. 5 (FucT RNAi design) and FIG. 33 (XylT RNAidesign).

Independent expression cassettes containing promoter, gene of interest,and Nos terminator were created for the optimized mAbI H and L chainsand the single-gene or chimeric RNAi. Expression cassettes were clonedinto a modification of the Agrobacterium binary vector pBMSP3 (obtainedfrom Dr. Stan Gelvin, Purdue University) with the appropriaterestriction sites. Depending upon the expression cassette, the L chainwas fused to either the modified chimeric octopine and mannopinesynthase promoter with Lemna gibba 5′ RbcS leader (mAbI01, FIG. 14) orthe high expression, constitutive Lemna minor polyubiquitin promoter(LmUbq) (mAbI04, FIG. 12; mAbI05; FIG. 13). The H-chain was fused to themodified chimeric octopine and mannopine synthase promoter with Lemnagibba 5′ RbcS leader (mAbI04, mAbI05, and mAbI01). The chimeric RNAicassette, taken from plasmid XF02 in T7-4, was fused to the highexpression, constitutive Spirodela polyrhiza polyubiquitin promoter(SpUbq). The single-gene RNAi cassette for expression of the FucTinhibitory sequence was driven by the SpUbq promoter; and the singlegene RNAi cassette for expression of the XylT inhibitory sequence wasdriven by an operably linked expression control element comprising theLemna aequinoctialis ubiquitin promoter plus 5′ UTR (LaUbq promoter).The H, L, and chimeric RNAi expression cassettes were cloned into themodified pBMSP3 binary vector in tandem orientation creating plasmidmAbI04. The H, L, and single-gene RNAi expression cassettes targetingFucT and XylT expression were cloned into the modified pBMSP3 binaryvector creating plasmid mAbI05. The H and L expression cassettes werecloned into the modified pBMSP3 binary vector creating plasmid mAbI01.

Though any transformation protocol can be used as noted herein above, insome embodiments, the transformation protocol was as follows. UsingAgrobacterium tumefaciens C58Z707, a disarmed, broad host range C58strain (Hepburn et al. (1985) J. Gen. Microbiol. 131:2961-2969),transgenic plants representing individual clonal lines were generatedfrom rapidly growing Lemna minor nodules according to the procedure ofYamamoto et al. (2001) In Vitro Cell Dev. Biol. Plant 37:349-353. Fortransgenic screening, individual clonal lines were preconditioned for 1week at 150 to 200 μmol m-2s-2 in vented plant growth vessels containingSH media (Schenk and Hildebrandt (1972) Can. J. Botany 50:199-204)without sucrose. Fifteen to twenty preconditioned fronds were thenplaced into vented containers containing fresh SH media, and allowed togrow for two weeks. Tissue and media samples from each line were frozenand stored at −70° C.

A MALDI-TOF assay was developed to measure L. minorβ-1,2-xylosyltransferase (XylT) and α-1,3-fucosyltransferase (FucT)activities (Example 3 below).

FIGS. 15-17 represent primary screening data for the XF02, mAbII04, andmAbI05 plants lines using the aforementioned assay. In this assay, WT(wild-type) represents the FucT and XylT activity in wild-type plantswhile BWT (boiled wild-type) represents their activity in boiled plantextracts. Boiled wild-type (BWT) plant extracts are representative ofplant material in which FucT and XylT activity has been deactivated.This data set shows that several plant lines from each construct have areduced level of FucT and XylT activity compared with wild-type plantlines (WT) and a comparable level of activity with boiled wild-typesamples (BWT).

Specifically, primary screening data for transgenic RNAi L. minor plantlines comprising the XF02 construct of FIG. 10 are shown in FIGS. 15 and16. The XF02 construct expresses a chimeric RNAi molecule that targetsexpression of both the L. minor FucT and XylT proteins, including thevarious isoforms of the respective proteins.

FIG. 17 shows primary screening data for transgenic RNAi L. minor plantlines comprising the mAbI04 construct of FIG. 12 and mAbI05 construct ofFIG. 13.

Example 3 MALDI-TOF Assay for N-glycan β-1,2-xylosyltransferase (XylT)and α-1,3-fucosyltransferase (FucT) Activity

The following modified MALDI-TOF assay was used to determine XylT andFucT activity in the transgenic plants described in Example 2 above.

Materials

-   HOMOGENIZATION BUFFER: 50 mM HEPES, pH 7.5, 0.25 M sucrose, 2 mM    EDTA, 1 mM DTT.-   REACTION BUFFER: 0.1 M Mes, pH 7.0, 10 mM MnCl₂, 0.1% (v/v) Triton    X-100.-   URIDINE-5′-DIPHOSPHO-D-XYLOSE (UDP-Xyl)-   GUANOSINE-5′-DIPHOSPHO-L-FUCOSE (GDP-Fuc)-   N-ACETYLGLUCOSAMINE-   POLYETHYLENE GLYCOL (PEG) MIXTURE 1000-3000 (10 mg/mL PEG 1000,    2000, and 3000 (4:5:6 ratio) mixed 4:1 with 2 mg/mL sodium iodide).-   [Glu¹]-FIBRINOPEPTIDE B (GFP), HUMAN (1 pmol/μL in water)-   DABSYLATED, TETRAPEPTIDE, N-GLYCAN ACCEPTOR (EMD Biosciences)-   CHCA (α-CYANO-4-HYDROXYCINNAMIC ACID) MATRIX (10 mg in 50% [v/v]    acetonitrile, 0.05% [v/v] trifluoroacetic acid).    Microsome Preparation

L. minor tissue (100 mg) was ground in 1 mL of cold homogenizationbuffer in a bead mill at 5× speed for 40 s. The homogenate was spun at1,000 g for 5 min, 4° C. The supernatant was removed and spun at 18,000g for 2 h, 4° C. The supernatant was then discarded. The pellet wasresuspended in 20 μL of cold reaction buffer and kept on ice or storedat −80° C. until use.

Reaction Conditions

The reaction mix contains 125 mM N-acetylglucosamine, 6.25 mM UDP-Xyl,6.25 mM GDP-Fuc, 12.5 mM MnCl₂, and 1.5 nmol of dabsylated, tetrapeptideN-glycan acceptor. Microsomes (4 μL) were added to the reaction mix tostart the reaction. The reaction was incubated for 30 min at roomtemperature, and 90 min at 37° C. The reaction was terminated bycentrifugation at 18,000 g for 1 min and incubation at 4° C.

MALDI-TOF Analysis

A portion of the supernatant from each reaction (0.5 μL) was mixed with0.5 μL of CHCA matrix on a MALDI target plate and allowed to dry. TheMALDI instrument was set to reflectron positive ion mode and calibratedwith PEG 1000-3000. Combined MS spectra (˜200 shots) were taken from1500-2500 Da using 0.5 μmol GFP as the lock mass. Ion counts of thereference peak (m/z=2222.865) should be above 400. Ion counts of theXylT and FucT products (m/z=2192.854 and 2206.870, respectively) werenormalized to the reference peak and the protein concentration of themicrosome fraction.

Example 4 Effect of RNAi Inhibition of Expression of L. minor FucT andXylT on Glycosylation Profile of Monoclonal Antibodies

Monoclonal antibodies produced by wild-type (i.e., FucT and XylTexpression not silenced) L. minor comprising the mAbI01 construct (seeFIG. 14) and L. minor lines transgenic for the mAbI04 construct (seeFIG. 12) or mAbI05 construct (see FIG. 13) were analyzed for theirN-glycosylation profile. The following procedures were used.

Purification of mAb from Lemna

Plant tissue was homogenized with 50 mM Sodium Phosphate, 0.3M SodiumChloride, and 10 mM EDTA at pH 7.2 using a Silverson High Shear Mixer ata tissue:buffer ratio of 1:8. The homogenate was acidified to pH 4.5with 1M Citric Acid, and centrifuged at 7,500×g for 30 minutes at 4° C.The supernatant was filtered through a 0.22 μm filter and loadeddirectly on mAbSelect SuRe resin (GE Healthcare) equilibrated with asolution containing 50 mM Sodium Phosphate, 0.3M Sodium Chloride, and 10mM EDTA, pH 7.2. After loading, the column was washed to baseline withthe equilibration buffer followed by an intermediate wash with 5 columnvolumes of 0.1M Sodium Acetate, pH 5.0, and finally, bound antibody waseluted with 10 column volumes of 0.1M Sodium Acetate, pH 3.0. The eluatewas immediately neutralized with 2M Tris base.

Purification of N-linked Glycans

Protein A-purified monoclonal antibodies (1 mg) from wild-type and RNAiL. minor plant lines were dialyzed extensively against water andlyophilized to dryness. Samples were resuspended in 100 μL of 5% (v/v)formic acid, brought to 0.05 mg/mL pepsin, and incubated at 37° C.overnight. The samples were heat inactivated at 95° C. for 10 min anddried. Pepsin digests were resuspended in 100 μL of 100 mM sodiumacetate, pH 5.0 and incubated with 1 mU of N-glycosidase A at 37° C.overnight. The released N-glycans were isolated using 4 cc CarbographSPE columns according to Packer et al. (1998) Glycoconj. J. 15: 737-747,and dried.

Dried N-glycans were further purified using 1 cc Waters Oasis MCXcartridges. Columns were prepared by washing with 3 column volumes ofmethanol followed by 3 column volumes of 5% (v/v) formic acid.N-glycans, resuspended in 1 mL of 5% (v/v) formic acid, were loaded ontothe prepared columns. The unbound fraction as well as 2 additionalcolumn volume washes of 5% (v/v) formic acid were collected, pooled anddried.

Derivatization of Oligosaccharides with 2-aminobenzoic acid (2-AA)

Purified N-glycans or maltooligosaccharides were labeled with 2-AA andpurified using 1 cc Waters Oasis HLB cartridges according to Anumula andDhume (1998) Glycobiology 8: 685-694. Labeled N-glycans andmaltooligosaccharides were resuspended in 50 μL of water and analyzed byMALDI-TOF MS and normal phase (NP) HPLC-QTOF MS.

MALDI-TOF Mass Spectrometry

MALDI-TOF MS was conducted using a Waters MALDI Micro MX (Millford,Mass.). 2-AA labeled N-glycans (0.5 μL) were properly diluted withwater, mixed with 0.5 μL of 10 mg/mL DHB matrix in 70% (v/v)acetonitrile, spotted onto a target plate and analyzed in negativereflectron mode.

NP-HPLC-Q-TOF MS Analysis of 2-AA Labeled N-glycans

2-AA labeled N-glycans or maltooligosaccharides were brought to 80%(v/v) acetonitrile and separated on a Waters 2695 HPLC system fittedwith a TSK-Gel Amide-80 (2 mm×25 cm, 5 μm) column (Tosoh Biosciences,Montgomeryville, Pa.). 2-AA labeled carbohydrates were detected andanalyzed by fluorescence (230 nm excitation, 425 nm emission) using aWaters 2475 fluorescence detector and a Waters Q-TOF API USquadropole-time of flight (Q-TOF) mass spectrometer (Millford, Mass.)fitted in-line with the HPLC system.

Separations were conducted at 0.2 mL/min, 40° C., using 10 mM ammoniumacetate, pH 7.3 (solvent A) and 10 mM ammonium acetate, pH 7.3, 80%(v/v) acetonitrile (solvent B). Sample elution was carried out at 0% Aisocratic for 5 min, followed by a linear increase to 10% A at 8 min,and a linear increase to 30% A at 48 min. The column was washed with100% A for 15 min and equilibrated at 0% A for 15 min prior to the nextinjection.

Q-TOF analysis was conducted in negative ion mode with source anddesolvation temperatures of 100° C. and 300° C., respectively, andcapillary and cone voltages of 2,100 and 30 V, respectively. Massspectra shown are the result of combining ≧50 individual scans perlabeled N-glycan.

RP-HPLC-Q-TOF MS Analysis of Intact IgG

Protein A purified IgG's (50 μg) were desalted using the Waters 2695HPLC system fitted with a Poros R1-10 column (2 mm×30 mm; AppliedBiosystems). IgG's were detected and analyzed using a Waters 2487 dualwavelength UV detector (280 nm) and the Waters Q-TOF API US. Separationswere conducted at 0.15 mL/min, 60° C., using 0.05% (v/v) trifluoroaceticacid (TFA; solvent A) and 0.05% (v/v) TFA, 80% (v/v) acetonitrile(solvent B). Sample elution was carried out using a linear increase from30 to 50% B for 5 min, an increase to 80% B for 5 min. The solvent ratioremained at 80% B for an additional 4 min, followed by a wash with 100%B for 1 min and equilibration of the column with 30% B for 15 min priorto the next run.

Q-TOF analysis was conducted in positive ion mode with source anddesolvation temperatures of 100° C. and 300° C., respectively, andcapillary and cone voltages of 3.0 and 60 V, respectively. Data are theresult of combining ≧100 individual scans and deconvolution to theparent mass spectrum using MaxEnt 1.

See also Triguero et al. (2005) Plant Biotechnol. J. 3: 449-457;Takahashi et al. (1998) Anal. Biochem. 255: 183-187; Dillon et al.(2004) J. Chromatogr. A. 1053: 299-305.

Results

FIG. 18 shows the structure and molecular weight of derivatizedwild-type L. minor monoclonal antibody N-glycans.

FIG. 19 shows that the wild-type mAbI01 construct (shown in FIG. 15)providing for expression of the mAbI monoclonal IgG1 antibody in L.minor, without RNAi suppression of L. minor FucT and XylT, produces anN-glycosylation profile with three major N-glycan species, including onespecies having the β1,2-linked xylose and one species having both theβ1,2-linked xylose and core α1,3-linked fucose residues; this profile isconfirmed with liquid chromatography mass spectrometry (LC-MS) (FIG. 20)and MALDI (FIG. 21) analysis.

FIG. 22 shows an overlay of the relative amounts of the various N-glycanspecies of the mAbI produced in the wild-type L. minor line comprisingthe mAbI01 construct (no suppression of FucT or XylT) and in the twotransgenic L. minor lines comprising the mAbI04 construct of FIG. 12.Note the enrichment of the GnGn (i.e., G0) glycan species, with noβ1,2-linked xylose or core α1,3-linked fucose residues attached, and theabsence of the species having the β1,2-linked xylose or both theβ1,2-linked xylose and core α1,3-linked fucose residues. This profile isconfirmed with mass spec (LC-MS) (FIG. 23) and MALDI (FIG. 24) analysis.

FIG. 25 shows intact mass analysis of the mAbI compositions produced inwild-type L. minor (line 20) comprising the mAbI01 construct. When XylTand FucT expression are not suppressed in L. minor, the recombinantlyproduced mAbI composition is heterogeneous, comprising at least 9different glycoforms, with the G0XF³ glycoform being the predominatespecies present. Note the very minor peak representing the G0 glycoform.

FIG. 26 shows intact mass analysis of the mAbI compositions produced intransgenic L. minor (line 15) comprising the mAbI04 construct of FIG.12. When XylT and FucT expression are suppressed in L. minor using thischimeric RNAi construct, the intact mAbI composition is substantiallyhomogeneous for G0 N-glycans, with only trace amounts of precursorN-glycans present (represented by the GnM and MGn precursor glycanspecies). In addition, the mAbI composition is substantially homogeneousfor the G0 glycoform, wherein both glycosylation sites are occupied bythe G0 N-glycan species, with three minor peaks reflecting trace amountsof precursor glycoforms (one peak showing mAbI having an Fc regionwherein the C_(H)2 domain of one heavy chain has a G0 glycan speciesattached to Asn 297, and the C_(H)2 domain of the other heavy chain isunglycosylated; another peak showing mAbI having an Fc region whereinthe C_(H)2 domain of one heavy chain has a G0 glycan species attached toAsn 297, and the C_(H)2 domain of the other heavy chain has the GnM orMGn precursor glycan attached to Asn 297; and another peak showing mAbIhaving an Fc region wherein the Asn 297 glycosylation site on each ofthe C_(H)2 domains has a G0 glycan species attached, with a third G0glycan species attached to an additional glycosylation site within themAbI structure).

FIG. 27 shows intact mass analysis of the mAbI compositions produced intransgenic L. minor (line 72) comprising the mAbI05 construct of FIG.13. When XylT and FucT expression are suppressed in L. minor using thisconstruct, the intact mAbI composition is substantially homogeneous forG0 N-glycans, with only trace amounts of precursor N-glycan speciespresent (represented by the GnM and MGn precursor glycan species). Inaddition, the mAbI composition is substantially homogeneous (at least90%) for the G0 glycoform, with the same three minor peaks reflectingprecursor glycoforms as obtained with the mAbI04 construct.

The receptor binding activity of the mAbI produced in the wild-typeLemna lines comprising the mAbI01 construct (i.e., without inhibition ofXylT and FucT expression) and the transgenic Lemna lines comprising themAbI04 or mAbI05 construct (i.e., with XylT and FucT expressioninhibited) was compared to the receptor binding activity of the mAbIproduced in mammalian cell lines (CHO and SP2/0).

Binding to FcFcγRIIIa on freshly isolated human NK cells was assessedfor the various mAbI products. Control data collected for CHO-derivedmAbI and SP2/0-derived mAbI is shown in FIG. 35. Test data collected forwild-type Lemna-produced mAbI having the normal plant N-glycan profileare designated as mAbI01-15 and mabI01-20, wherein the mAbI product hasN-linked glycans that include α(1,3)-fucose residues (see FIGS. 36 and37). Test data collected for transgenic Lemna-derived mAbI having anoptimized N-glycan profile (OPT) obtained with gene-silencing RNAiconstructs that target expression of α1,3-fucosyltransferase aredesignated as mAbI05-72, mAbI05-74, mAbI04-24, and mAbI04-15 (see FIGS.36 and 37), wherein the mAbI product has N-linked glycans that aredevoid of α(1,3)-fucose residues. Data comparing binding efficacy ofmAbI01-15, mAbI01-20, mAbI SP2/0, mAbI04-15, mAbI04-24, mAbI05-72, andmAbI05-74 to recombinant mouse FcγRIV, a receptor that is sensitive toIgG fucose levels and which served as a surrogate for human FcγRIIIa, isshown in FIG. 38.

These data demonstrate that the transgenic Lemna-derived mAbI producthaving the optimized glycan profile (OPT) shows enhanced binding toFcγRIIIa on freshly isolated human NK cells (enhanced about 20 to50-fold) as well as enhanced binding to recombinant mouse FcγRIV(enhanced about 10-fold) as compared to the wild-type Lemna-derived mAbIproduct.

Example 6 Production of Anti-CD30 Monoclonal Antibody Having ImprovedReceptor Binding and Increased ADCC Activity

This example outlines the expression of human anti-CD30 mAbs in Lemna.Optimization of anti-CD30 mAb glycosylation was accomplished byco-expression with an RNAi construct targeting the endogenous expressionof α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT)genes in a manner similar to that noted in the examples above for mAbI.The resultant anti-CD30 mAb produced in Lemna having its nativeglycosylation machinery engineered to suppress FucT and XylT expressioncontained a single major N-glycan species without any trace ofplant-specific N-glycans. In addition to the N-glycan homogeneity,glyco-optimized anti-CD30 mAbs were also shown to have enhancedantibody-dependent cell-mediated cytotoxicity (ADCC) and effector cellreceptor binding activity when compared to CHO-expressed anti-CD30 mAbs.

Methods

Strains and Reagents

Novablue competent Escherichia coli cells were used for all recombinantDNA work (EMD Biosciences, San Diego, Calif.). Restriction endonucleasesand DNA modification enzymes were obtained from New England Biolabs(Ipswich, Mass.). Oligonucleotides were obtained from Integrated DNAtechnologies (Coralville, Iowa). Waters Oasis HLB and MCX columns (1cc), 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid(CHCA) were from Waters Corporation (Milford, Mass.). Purifieddabsylated, tetrapeptide, GnGn N-glycan acceptors (GnGn-dabsyl-peptide)and N-glycosidase A were from EMD Biosciences. Carbograph SPE columns (4cc) were from Grace Davidson Discovery Sciences (Deerfield, Ill.).Uridine-5′-diphospho-D-xylose (UDP-Xyl) was purchased from CarbosourceServices (Athens, Ga.). Acetonitrile (Optima grade) was from FisherScientific (Summerville, N.Y.). Ammonium acetate was from MPBiochemicals (Irvine, Calif.). Maltooligosaccharides (MD6-1) were fromV-Labs Inc. (Covington, Calif.). Monosaccharide standards were fromDionex (Sunnyvale, Calif.). BATDA(bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxylate) andEuropium solution were from Perkin-Elmer (Wellesley, Mass.).Guanosine-5′-diphospho-L-fucose (GDP-Fuc), N-acetylglucosamine (GlcNAc),2-aminobenzoic acid (2-AA) and all other materials were from Sigma (St.Louis, Mo.).

Construction of mAb and RNAi Expression Vectors

The heavy (H) and light (L) chain variable region cDNA sequences offully human mAb 1 kappa antibody MDX-060 derived from a transgenicMedarex HuMAb-Mouse® (Borchmann et al. (2003) Blood 102:3737-3742) weredetermined and the full length MDX-060 human mAb 1 kappa antibody wasproduced recombinantly by a Chinese hamster ovary cell line, CHO DG44(Urlaub et al. (1986) Cell Mol. Genet. 12:555-566), using standardtechniques. Optimized genes for H and L chains were designed to haveLemna-preferred codon usage (63%-67% GC content) and contain the riceα-amylase signal sequence (GenBank M24286) fused to the 5′ end of theircoding sequences. Restriction endonuclease sites were added for cloninginto Agrobacterium binary vectors (EcoRI (5′)/SacI (3′), H-chain) and(SalI (5′)/HindIII (3′), L-chain). Synthetic genes were constructed andprovided by Picoscript (Houston, Tex.).

A chimeric hairpin RNA (see FIG. 34) was designed to target silencing ofendogenous Lemna genes encoding α-1,3-fucosyltransferase (based on thecoding sequence for L. minor FucT isoform #1, set forth in SEQ ID NO:2;see also GenBank DQ789145) and β-1,2-xylosyltransferase (based on thecoding sequence for L. minor XylT isoform #2, nt 1-1275 of SEQ ID NO:19;set forth in SEQ ID NO:20; see also GenBank DQ789146). The chimericFucT+XylT hairpin RNA was designed to have 602 bp of double strandedFucT sequence, 626 bp of double stranded XylT sequence, and 500 bp ofspacer sequence. The sense strand portion of the hairpin RNA cassetteencompasses the FucT forward fragment (nt 12-613 of SEQ ID NO:2;equivalent to nt 254-855 of SEQ ID NO:1) and XylT forward fragment (nt1-626 of SEQ ID NO:19), a spacer sequence (nt 627-1126 of SEQ ID NO:19).The antisense strand portion of the hairpin RNA encompasses the XylTreverse fragment (antisense version of nt 1-626 SEQ ID NO:19) and FucTreverse fragment (antisense version of nt 12-613 of SEQ ID NO:2 or nt254-855 of SEQ ID NO:1). The chimeric hairpin RNA was constructed by PCRamplifying FucT and XylT forward and reverse gene fragments from Lemnaminor (8627) cDNA and sequentially cloning them into pT7blue (EMDBiosciences) creating plasmid XF02 in T7-4. The FucT forward genefragment was amplified with DNA primers BLX 686(5′-ATGGTCGACTGCTGCTGGTGCTC TCAAC-3′) (SEQ ID NO:22) and BLX690(5′-ATGTCTAGAATG CAGCAGCAAGTGCACC-3′) (SEQ ID NO:23) producing a 620 bpproduct with terminal SalI (5′) and XbaI (3′) cloning sites. The XylTforward gene fragment was amplified with DNA primers BLX 700(5′-ATGACTAGTTGC GAAGCCTACTTCGGCAACAGC3′) (SEQ ID NO:24) and BLX694(5′-ATGGGATCCGAATCTCAAGA ACAACTGTCG-3′) (SEQ ID NO:25) producing a 1144bp product with terminal SpeI (5′) and BamHI (3′) cloning sites. TheXylT reverse gene fragment was amplified with DNA primers BLX 695(5′-ATGGGTACCTGCGAAGCCTACTTCGGCAA CAGC-3′) (SEQ ID NO:26) and BLX696(5′-ATGGGA TCCACTGGCTGGGAGAAGTTCTT-3′) (SEQ ID NO:27) producing a 644 bpproduct with terminal BamHI (5′) and KpnI (3′) cloning sites. The FucTreverse gene fragment was amplified with DNA primers BLX 691(5′-ATGGAGCTCTGCTGCTGGTGCT CTCAAC-3′) (SEQ ID NO:28) and BLX692(5′-ATGGGTACCATGCAGCAGCAAGTGCACC-3′) (SEQ ID NO:29) producing a 620 bpproduct with terminal KpnI (5′) and SacI (3′) cloning sites.

Independent expression cassettes containing promoter, gene of interest,and Nos terminator were created for the optimized MDX-060H and L chainsand the chimeric RNAi. Expression cassettes were cloned into amodification of the Agrobacterium binary vector pBMSP3 (obtained fromDr. Stan Gelvin, Purdue University) with the appropriate restrictionsites. The H chain was fused to the modified chimeric octopine andmannopine synthase promoter with Lemna gibba 5′ RbcS leader (Gasdaska etal. (2003) Bioprocessing J. 50-56). The L-chain was fused to the highexpression, constitutive Lemna minor polyubiquitin promoter (LmUbq). Thechimeric RNAi cassette, taken from plasmid XF02 in T7-4, was fused tothe high expression, constitutive Spirodela polyrhiza polyubiquitinpromoter (SpUbq). The three expression cassettes were cloned into themodified pBMSP3 binary vector in tandem orientation creating plasmidMDXA04.

Transformation and Plant Line Screening

Using Agrobacterium tumefaciens C58Z707 (Hepburn et al. (1985) J. Gen.Microbiol. 131:2961-2969), transgenic plants representing individualclonal lines were generated from rapidly growing Lemna minor nodulesaccording to the procedure of Yamamoto et al. (Yamamoto et al. (2001) Invitro Cell. Dev. Biol. 37). For transgenic screening, individual clonallines were preconditioned for 1 week at 150 to 200 μmol m⁻ ²s⁻² invented plant growth vessels containing SH media (Schenk and Hildenbrandt(1972) Can. J. Botany 50:199-204) without sucrose. Fifteen to twentypreconditioned fronds were then placed into vented containers containingfresh SH media, and allowed to grow for two weeks. Tissue and mediasamples from each line were frozen and stored at −70° C.

ELISA Analysis of mAb Produced in Lemna

Lemna tissue (100 mg) was homogenized using a FastPrep FP120 bead mill(Thermo Electron Corporation). Supernatants were diluted to 1 μg/mL andassayed using the IgG Quantitation ELISA kit (Bethyl Laboratories). Forthe assay, microtiter plates were coated with goat anti-human IgG at aconcentration of 10 μg/mL, and mAb was detected by horseradishperoxidase (HRP)-conjugated goat anti-human IgG diluted 1:100,000.Standard curves were created with Human Reference IgG supplied with theELISA kit. The sensitivity of the ELISA was 7.8 ng/mL. All samples wereanalyzed in duplicate.

Preparation of Lemna Microsomal Membranes and Assaying for Coreβ-1,2-xylosyltransferase and α-1,3-fucosyltransferase Activities

Lemna tissue (100 mg) from each line was homogenized in 1 mL of coldhomogenization buffer (50 mM4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 0.25M sucrose, 2 mM ethylenediaminetetraacetic acid [EDTA], 1 mM1,4-dithiothreitol [DTT]) for 40 s in a FastPrep FP120 bead mill (ThermoElectron Corporation, Waltham, Mass.). The homogenate was centrifuged at1,000 g for 5 min at 4° C. The supernatant was removed and centrifugedat 18,000 g for 90 min at 4° C. The resulting pellet was resuspended in20 μL of cold reaction buffer (0.1 M 2-[4-morpholino]ethanesulfonic acid[Mes], pH 7.0, 0.1% [v/v] Triton X-100, 10 mM MnCl₂) and kept on ice orstored at −80° C. until use.

Core β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activitieswere measured simultaneously in 4 μL of microsomal membranes preparedfrom each RNAi line by incubating with 125 mM GlcNAc, 6.25 mM UDP-Xyl,6.25 mM GDP-Fuc, 12.5 mM MnCl₂, and 1.5 nmol of GnGn-dabsyl-peptideacceptor for 2 h at 37° C. as described previously (Leiter et al. (1999)J. Biol. Chem. 274:21830-21839). The reaction was terminated by a briefcentrifugation and incubation at 4° C. and the products were analyzed bypositive reflectron mode MALDI-TOF MS.

Purification of MDX-060 LEX and LEX^(Opt) mAbs

Plant tissue was homogenized with 50 mM sodium phosphate, 0.3 M sodiumchloride, and 10 mM EDTA, pH 7.2 using a Silverson high shear mixer. Thehomogenate was acidified to pH 4.5 with 1 M citric acid, and centrifugedat 7,500 g for 30 min at 4° C. The pH of the supernatant was adjusted topH 7.2 with 2 M Tris, prior to filtration using 0.22 μm filters. Thematerial was loaded directly on mAbSelect SuRe protein A resin (GEHealthcare) equilibrated with a solution containing 50 mM sodiumphosphate, 0.3 M sodium chloride, and 10 mM EDTA, pH 7.2. After loading,the column was washed to baseline with the equilibration buffer followedby an intermediate wash with 5 column volumes of 0.1 M sodium acetate,pH 5.0. Bound antibody was eluted with 10 column volumes of 0.1 M sodiumacetate, pH 3.0. The protein A eluate was immediately neutralized with 2M 2-amino-2-[hydroxymethyl]-1,3-propanediol (Tris). For aggregateremoval, the protein A eluate was adjusted to pH 5.5 and applied to aceramic hydroxyapatite type I (Bio-Rad) column equilibrated with 25 mMsodium phosphate, pH 5.5. After washing the column with 5 column volumesof equilibration buffer, the antibody was eluted in a singlestep-elution using 0.25 M sodium phosphate, pH 5.5. Fractions containingantibody by A₂₈₀ were pooled and stored at −80° C.

Tissue extract and protein A flow through samples were prepared forSDS-PAGE under reducing and non-reducing conditions by addition of 2×SDSsample buffer ±5% [v/v] 2-mercaptoethanol. Protein A eluate andhydroxyapatite eluate samples were diluted to a protein concentration of0.5 mg/mL followed by addition of 2×SDS sample buffer ±5% [v/v]2-mercaptoethanol. Samples were incubated at 95° C. for 2 minutes priorto electrophoresis using 4-20% Tris-Glycine gradient gels (Invitrogen,Carlsbad, Calif.). Mark12 Molecular weight markers (Invitrogen) and aMDX-060 reference standard were included on the gels. Gels were stainedwith Colloidal Blue stain.

Purification of N-Linked Glycans

Protein A purified monoclonal antibodies (1 mg) from wild-type and RNAiLemna plant lines were dialyzed extensively against water andlyophilized to dryness. Samples were resuspended in 100 μL of 5% (v/v)formic acid, brought to 0.05 mg/ml pepsin, and incubated at 37° C.overnight. The samples were heat inactivated at 95° C. for 15 min anddried. Pepsin digests were resuspended in 100 μL of 100 mM sodiumacetate, pH 5.0 and incubated with 1 mU of N-glycosidase A at 37° C.overnight. The released N-glycans were isolated using 4 cc CarbographSPE columns (Packer et al. (1998) Glycoconj. J. 19:737-747) and dried.

Dried N-glycans were further purified using 1 cc Waters Oasis MCXcartridges. Columns were prepared by washing with 3 column volumes ofmethanol followed by 3 column volumes of 5% (v/v) formic acid.N-glycans, resuspended in 1 mL of 5% (v/v) formic acid, were loaded ontothe prepared columns. The unbound fraction as well as 2 additionalcolumn volume washes of 5% (v/v) formic acid were collected, pooled, anddried.

Derivatization of Oligosaccharides with 2-aminobenzoic acid (2-AA)

Purified N-glycans or maltooligosaccharides were labeled with 2-AA andpurified using 1 cc Waters Oasis HLB cartridges according to Anumula andDhume, 1998 (Anumula and Dhume (1998) Glycobiology 8:685-694). LabeledN-glycans and maltooligosaccharides were resuspended in 50 μL of waterand analyzed by negative mode MALDI-TOF MS and NP-HPLC-QTOF MS.

MALDI-TOF Mass Spectrometry

MALDI-TOF MS was conducted using a Waters MALDI Micro MX (Millford,Mass.). Analysis of β-1,2-xylosyltransferase/α-1,3-fucosyltransferasereaction products was conducted by mixing 0.5 μL of each reactionsupernatant with 0.5 μL of 10 mg/mL CHCA in 0.05% (v/v) TFA, 50% (v/v)acetonitrile on a target plate. Xylosylated ([M+H]⁺=2192.85 Da) orfucosylated ([M+H]⁺=2206.87 Da) GnGn-dabsyl-peptide products weredetected in positive reflectron mode. Ion counts of 200 combined spectrafrom each sample were normalized against that of β-1,4-galactosylated,GnGn-dabsyl-peptide ([M+H]⁺=2222.87 Da) present as a contaminant (<5%)in the original GnGn-dabsyl-peptide mixture from EMD Biosciences.

2-AA labeled N-glycans or maltooligosaccharides (0.5 μL) were dilutedwith water, mixed with 0.5 μL of 10 mg/ml DHB matrix in 70% (v/v)acetonitrile, spotted onto a target plate and analyzed in negativereflectron mode.

NP-HPLC-Q-TOF MS Analysis of 2-AA Labeled N-Glycans

2-AA labeled N-glycans or maltooligosaccharides were brought to 80%(v/v) acetonitrile and separated on a Waters 2695 HPLC system fittedwith a TSK-Gel Amide-80 (2 mm×25 cm, 5 μm) column (Tosoh Biosciences,Montgomeryville, Pa.). 2-AA labeled carbohydrates were detected andanalyzed using a Waters 2475 fluorescence detector (230 nm excitation,425 nm emission) and a Waters Q-TOF API US quadropole-time of flight(QTOF) mass spectrometer fitted on-line with the HPLC system.

Separations were conducted at 0.2 mL/min, 40° C., using 10 mM ammoniumacetate, pH 7.3 (solvent A) and 10 mM ammonium acetate, pH 7.3, 80%(v/v) acetonitrile (solvent B). Sample elution was carried out at 0% Aisocratic for 5 min, followed by a linear increase to 10% A at 8 min,and a linear increase to 30% A at 48 min. The column was washed with100% A for 15 min and equilibrated at 0% A for 15 min prior to the nextinjection.

QTOF analysis was conducted in negative ion mode with source anddesolvation temperatures of 100° C. and 300° C., respectively, andcapillary and cone voltages of 2,100 and 30 V, respectively. Massspectra shown are the result of combining ≧40 individual scans perlabeled N-glycan.

Monosaccharide Analysis by HPAEC-PAD

mAb samples (200 μg) were subjected to acid hydrolysis using 2 N TFA at100° C. for 3 h. Samples were dried by vacuum centrifugation at ambienttemperature and reconstituted in 150 μL water prior to analysis byHPAE-PAD (Dionex). An aliquot (25 μL) of the reconstituted sample wasapplied to a CarboPac PA10 column (4×250 mm) with a pre-column AminoTrap (Dionex). Separation of monosaccharides was accomplished with amobile phase of 3.5 mM KOH, using an EG40 eluent generator.Monosaccharide peak identity and relative abundance were determinedusing monosaccharide standards.

Thermal Stability of mAb

A MicroCal (Northampton, Mass.) VP-Capillary differential scanningcalorimetry (DSC) instrument was used to determine thermal stability ofglycol-optimized and wild-type mAbs. Purified mAb samples were dialyzedin 20 mM NaH₂PO₄, pH 7.4, 150 mM NaCl (PBS) overnight. Thermaldenaturation data was collected by heating the samples at aconcentration of 300 μg/mL from 35 to 95° C. at a scan rate of 1° C./minusing PBS as the reference buffer. The feedback and gain were set tolow. The baseline-corrected and normalized data was fit to a non-2-statemodel using Origin v7.0 software.

FcR Binding Activity of mAb

The experiment was conducted using a BIACORE (Biacore AB, Uppsala,Sweden) instrument using surface plasmon resonance technology. mAbs, 2μg/mL, were captured on the antigen coated surface (recombinant humanCD30). Several concentrations of both the Val¹⁵⁸ and Phe¹⁵⁸ allotypes ofFcRγIIIa, starting from 6 μM, were flowed over the captured antibodiesfor 3 min. The binding signal as a function of FcRγIIIa was fit to aone-site binding model using GraphPad Prism (v4.01) software to obtainthe K_(D) values. HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA and0.005% (v/v) P20 at pH 7.4) was used throughout the experiment. Bindingof the mAbs to buffer or FcRγIIIa to blank surfaces were used asnegative controls.

Assay for Antigen Binding Affinity

CD30-expressing L540 cells (DSMZ Cell Culture Collection # ACC 72) wereused as antigen positive cells to assay for binding. Aliquots of 2×10⁵cells/well were incubated for 30 min at 4° C. with 100 μL of primaryantibody at the indicated concentrations. Cells were washed twice in PBSwith 2% (v/v) fetal bovine serum (FBS) before addition of goatanti-human mAb, FITC-labeled secondary antibody (Jackson ImmunoResearch,West Grove, Pa.) at 1:500 dilution in 100 μL/well for 30 min at 4° C.Cells were washed twice in PBS with 2% (v/v) FBS and assayed by flowcytometry using a FACS Calibur instrument (Becton Dickinson, FranklinLakes, N.J.). EC₅₀ values of MDX-060 CHO, LEX and LEX^(Opt) mAb bindingto CD30 on L540 cells were determined from binding curves utilizingGraphPad Prism 3.0 software.

ADCC Assay

Human peripheral blood mononuclear effector cells were purified fromheparinized whole blood by standard Ficoll-Paque separation. Cells(2×10⁶) were washed in PBS and sent for genotyping. The remainingeffector cells were then resuspended at 1×10⁶ cells/mL in RPMI 1640medium containing 10% (v/v) FBS and 50 U/mL of human IL-2 (ResearchDiagnostics, Concord, Mass.) and incubated overnight at 37° C. Theeffector cells were washed once in culture medium and resuspended at1×10⁷ cells/mL prior to use. L540 target cells at 1×10⁶ cells/mL in RPMI1640 medium containing 10% (v/v) FBS and 5 mM probenecid were labeledwith 20 μM BATDA(bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxylate) for 20 minat 37° C. Target cells were washed three times in PBS supplemented with20 mM HEPES and 5 mM probenecid, resuspended at 1×10⁵ cells/mL and addedto effector cells in 96-well plates (1×10⁴ target cells and 5×10⁵effector cells/well) at a final target to effector ratio of 1:50.Maximal release was obtained by incubation of target cells in 3% (v/v)Lysol and spontaneous release obtained by incubation in cell culturemedium alone. After 1 h incubation at 37° C., 20 μL of supernatant washarvested from each well and added to wells containing 180 μL ofEuropium solution. The reaction was read with a Perkin Elmer FusionAlpha TRF reader using a 400 μsec delay and 330/80, 620/10 excitationand emission filters respectively. The counts per second were plotted asa function of antibody concentration and the data was analyzed bynon-linear regression, sigmoidal dose response (variable slope) usingGraphPad Prism 3.0 software. The percent specific lysis was calculatedas: (experimental release−spontaneous release)/(maximalrelease−spontaneous release)×100. In all studies, human mAb1 isotypecontrol was included and compared to MDX-060 CHO, LEX, and LEX^(Opt)mAbs. Other controls included target and effector cells with no mAb,target cells with no effector cells and target and effector cells in thepresence of 3% (v/v) Lysol.

Results

Expression of MDX-060 mAb in the LEX System.

MDX-060 is an anti-CD30 antibody (formally known as 5F11) beingdeveloped for the treatment of Hodgkins lymphoma and anaplastic largecell lymphoma (Borchmann et al. (2003) Blood 102:3737-3742). Two binaryvectors were constructed for the expression of MDX-060 in the LEXsystem. Expression vector MDXA01 contained codon optimized genesencoding heavy (H) and light (L) chains of MDX-060 while vector MDXA04contained genes encoding H and L chains in addition to a chimericFucT/XylT RNAi gene (FIG. 39). Independent transgenic lines weregenerated for both the MDXA01 (165 lines) and MDXA04 (195 lines)expression vectors. For simplicity, MDXA01 derived mAbs (wild-typeN-glycosylation), and MDXA04 derived mAbs (containing the FucT/XylT RNAiconstruct) will be referred to as MDX-060 LEX and MDX-060 LEX^(Opt),respectively, in the discussions below.

Transgenic plant lines were first screened for mAb expression with anIgG ELISA. LEX^(Opt) lines with high levels of mAb expression wereassayed further for FucT and XylT activity. Transferase activities inthe majority of the high expressing MDX-060 LEX^(Opt) lines were reducedto levels of the negative control indicating effective silencing in themajority of the assayed lines (FIG. 40). MDX-060 LEX^(Opt) lines did notexhibit any morphological or growth differences compared to wild-typeLemna plants (data not shown).

Thermal stabilities of the MDX-060 CHO, LEX, and LEX^(Opt) mAbs weredetermined using differential scanning calorimetry (DSC). All three mAbsexhibited similar melting curve kinetics (data not shown) and meltingtransition point temperatures (Table 2 below), further demonstrating thestructural integrity of the Lemna-produced MDX-060 LEX and LEX^(Opt)mAbs compared to the MDX-060 CHO mAb. SDS-PAGE analysis undernon-reducing (FIG. 41A) and reducing conditions (FIG. 41B) showed thatmAbs from the MDX-060 LEX^(Opt) and MDX-060 CHO lines had similarprotein profiles with the mAb appearing as the major component in theprotein extract.

TABLE 2 Comparison of the thermal stabilities of MDX-060 CHO, MDX-060LEX, and glyco-optimized MDX-060 LEX^(Opt) mAbs by differential scanningcalorimetry (DSC). Antibody T_(m1) (° C.) T_(m2) (° C.) T_(m3) (° C.)MDX-060 CHO 72 75 84 MDX-060 LEX 71 75 84 MDX-060 LEX^(Opt) 72 76 84N-Glycan Structures of MDX-060 CHO, LEX, and LEX^(Opt) mAbs

The N-glycan profiles of recombinant MDX-060 CHO, MDX-060 LEX, andMDX-060 LEX^(opt) derived mAbs were determined by negative reflectronmode MALDI-TOF MS and normal phase (NP) HPLC-QTOF MS. The structures ofN-glycans referred to in the following discussion are shown in FIG. 53.

MALDI-TOF MS analysis of N-glycans from MDX-060 CHO lines indicated thepresence of four major N-glycans with m/z values corresponding to 2-AAlabeled GnGnF⁶ (nomenclature derived from http://www.proglycan.com),Man5, GnA_(iso)F⁶, and AAF⁶ (FIG. 42). NP-HPLC separated the GnA_(iso)F⁶N-glycan into its two isoforms (Gal attached to the α-1,6-Man orα-1,3-Man arm) bringing the total number of major N-glycans found onMDX-060 CHO to five (FIG. 43). MS/MS fragmentation of the peaks was notconducted to confirm the identity of each isoform; however, the higherabundance of the earlier peak suggested that Gal was attached to theα-1,6-Man arm of this N-glycan (Shinkawa et al. (2003) J. Biol. Chem.278:3466-3473; Zhu et al. (2005) Nat. Biotechnol. 23:1159-1169). On-linenegative mode QTOF MS analysis gave m/z values corresponding to doublycharged GnGnF⁶, Man5, GnA_(iso)F⁶ (both isoforms), and AAF⁶, confirmingthe MALDI-TOF MS results (Table 3 below). Peak integration of thefluorescent trace revealed that GnGnF⁶, Man5, AGnF⁶, GnAF⁶, and AAF⁶constituted 50.8, 2.5, 26.1, 10.7 and 6.8%, respectively, of the totalN-glycan structures from MDX-060 CHO. The remaining 3.1% of N-glycanswere found to be a mixture of GnGn, GnM_(iso)F⁶, GnM_(iso), and MM withno structure higher than 1.2% of the total (data not shown).

TABLE 3 Summary of observed MALDI-TOF and QTOF MS masses of the major2-AA labeled N-glycans from MDXA-060 mAbs produced by CHO cells (CHO),wild-type Lemna (LEX) or glyco-optimized Lemna lines expressing the RNAicontruct (LEX^(Opt)). Observed Observed Q- Proposed Theoretical m/zMALDI-TOF^(c) TOF^(c) % Peak N-glycan name^(a) Structure^(b) [M − H]⁻ [M− 2H]²⁻ [M − H]⁻ [M − 2H]²⁻ Area^(c) CHO GnGnF⁶-2AA

1582.590 790.7911 1582.455 790.7825 50.8 Man5-2AA

1354.479 676.7436 1354.392 676.7343 2.50 GnA_(iso)F⁶-2AA

1744.642 871.8175 1744.492 871.7970 36.8 AAF⁶-2AA

1906.695 952.8438 1906.567 952.8181 6.80 LEX GnGn-2AA

1436.532 717.7622 1436.549 717.7894 8.40 GnGnX-2AA

1568.574 783.7833 1568.581 783.8150 17.2 GnGnXF³-2AA

1714.632 856.8122 1714.615 856.853 67.4 LEX^(Opt) GnGn-2AA

1436.532 717.7622 1436.523 717.7993 95.8

MALDI-TOF MS analysis of wild-type MDX-060 LEX mAb revealed the presenceof three major species with m/z values corresponding to GnGnXF³, GnGnXand GnGn (FIG. 42). NP-HPLC-QTOF MS analysis showed three majorfluorescent peaks with m/z values corresponding to doubly chargedGnGnXF³, GnGnX and GnGn, again confirming the MALDI-TOF MS results (FIG.43; Table 3). Integration of the fluorescent peaks indicated thatGnGnXF³, GnGnX and GnGn constituted 67.4, 17.2 and 8.4%, respectively,of the total N-glycans derived from the MDX-060 LEX mAb. The remaining7% of N-glycans were determined to be a mixture of MM, GnM_(iso), MMXF³,GnGnF³, GnM_(iso)XF³, Man6, Man7, Gn(FA)_(iso)XF³, Man8 and Man9 with noN-glycan greater than 2% of the total. Similar results were seen withmAbs isolated from two independently transformed MDX-060 LEX lines (datanot shown). The simple array of N-glycans on LEX mAbs demonstrated hereprovides an amenable starting point for glyco-optimization.

In contrast to the MDX-060 LEX mAb, N-glycans from the MDX-060 LEX^(Opt)mAb possessed GnGn as the major N-glycan species by both MALDI-TOF MSand NP-HPLC-QTOF MS analysis (FIGS. 42 and 43; Table 3). GnGn comprised95.8% of the total N-glycans with the remaining 4.2% of N-glycansdetermined to be MM, GnM_(iso), GnA_(iso), Man6, Man7 and Man8 with noone structure greater than 1.2% of the total N-glycans. None of theLEX^(Opt) N-glycans contained fucose (Fuc) or xylose (Xyl). Theseresults demonstrated that co-expression of an RNAi construct targetingLemna FucT and XylT resulted in the complete elimination of Fuc andXyl-containing N-glycans from MDX-060 LEX^(Opt) mAbs and produced highlyhomogeneous mAb glycoforms. The same results were obtained for MDX-060LEX^(Opt) mAb harvested from an independent transgenic line (line 225)comprising the MDXA04 expression vector, at a different growth scale(300 g tissue for transgenic line 225 versus 1 g tissue for transgenicline 52, which produced the MDX-060 LEX^(Opt) mAb having the N-glycanprofile shown in FIGS. 42 and 43. Unlike mammalian cell culture systemswhere N-glycan heterogeneity can change with culture conditions, growthscale, and growth period (Kanda et al. (2006) Biotechnol. Bioeng.94:680-688), the glycan uniformity observed with LEX^(Opt) mAbs wasshown to be consistent under a variety of growth conditions and scales.

The absence of Fuc or Xyl on MDX-060 LEX^(Opt) mAb N-glycans was furtherconfirmed by monosaccharide analysis (Table 4 below). Monosaccharideswere released from MDX-060 CHO, LEX and LEX^(Opt) mAbs by acidhydrolysis and analyzed by high performance anion exchangechromatography (HPAEC) coupled to pulsed amperometric detection (PAD).The monosaccharide ratios for Man and GlcNAc residues were similar forCHO and wild-type LEX mAbs and correlated well with expected values. LEXmAbs were significantly decreased in Gal and Fuc content and had asignificant increase in Xyl when compared to CHO-derived mAbs.Monosaccharide analysis of Lemna derived mAbs revealed that while Fucand Xyl were present on wild-type LEX N-glycans, they were not detectedon LEX^(Opt) mAbs. Collectively, these results demonstrate thatco-expression of an RNAi construct targeting Lemna XylT and FucT resultsin the elimination of Fuc and Xyl-containing N-glycans from MDX-060LEX^(Opt) mAbs and produce highly homogeneous mAb glycoforms. Therobustness of this glyco-optimization strategy has been confirmed withmultiple independent plant lines expressing the MDX-060 LEX^(Opt) mAb aswell as with other mAbs expressed in the LEX System, for example, themAbI monoclonal antibody discussed in the examples herein above. Inthese subsequent transformations, glyco-optimized mAb expression levelsup to 6% of total soluble protein (TSP) have been obtained.

TABLE 4 Monosaccharides released from MDX-060 CHO, LEX and LEX^(Opt)mAbs by acid hydrolysis and analyzed by HPAEC-PAD. The monosaccharidecontent from each mAb was determined by normalizing against carbohydratecontrols. MDX-060 MDX-060 CHO MDX-060 LEX LEX^(Opt) Monosaccharide pmol(% total) pmol (% total) pmol (% total) Fuc 254 (20) 232 (13) 0 GlcNAc605 (47) 773 (45) 1,003 (67)  Gal 75 (6) 0 0 Man 355 (27) 491 (29) 501(33) Xyl 0 226 (13) 0 Total 1,289 (100)  1,722 (100)  1,504 (100) Functional Activity of MDX-060 CHO, LEX and LEX^(Opt) mAbs

Antigen binding properties of the MDX-060 CHO, MDX-060 LEX, and MDX-060LEX^(Opt) mAbs were determined using CD30 expressing L540 cells. Allthree mAbs had nearly identical binding curves (FIG. 43). EC₅₀concentrations were determined to be 0.180 μg/mL, 0.227 μg/mL, and 0.196μg/mL for MDX-060 CHO, LEX, and LEX^(Opt), respectively (FIG. 44),indicating that antigen binding for all three mAbs were similar.

Fc-receptor-mediated effector cell function has been shown to beimportant for the in vivo activity of many therapeutic mAbs. Since theFcR expressed on NK cells and macrophages responsible for ADCC activityis FcγRIIIa, binding of the various mAbs to this receptor was compared.FcR binding of CHO, LEX and LEX^(Opt) mAbs was determined by equilibriumbinding of the mAbs with effector cells expressing two different humanFcRγIIIa allotypes (Phe¹⁵⁸ or Val¹⁵⁸). MDX-060 LEX had a 1.7-foldincrease in FcRγIIIaPhe¹⁵⁸ and a 0.4-fold decrease in FcRγIIIaVal¹⁵⁸binding compared to the CHO-derived mAb, demonstrating that receptorbinding for CHO and LEX mAbs were similar. In contrast, LEX^(Opt) mAbshad a 27 and 15-fold higher affinity for FcRγIIIaPhe¹⁵⁸ andFcRγIIIaVal¹⁵⁸, respectively, than CHO mAbs (FIG. 45). These resultssuggested that RNAi silencing of the Lemna FucT and XylT activities inLEX^(Opt) lines produced mAbs with enhanced FcR binding.

ADCC activities of the CHO, LEX and LEX^(Opt) mAbs were determined byincubating mAbs with either homozygous (FcRγIIIaPhe¹⁵⁸) or heterozygous(FcRγIIIaPhe/Val¹⁵⁸) human effector cells and BATDA(bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxylate) labeledL540 target cells (FIG. 45). MDX-060 LEX mAbs (31%) had the same maximalpercent cell lysis as CHO mAbs (31%) using heterozygousFcRγIIIaPhe/Val¹⁵⁸ human effector cells (FIG. 46) with similar EC₅₀values. Maximal percent cell lysis for LEX mAbs (27%) was slightlyincreased compared to CHO mAbs (15%) using homozygous FcγRIIIaPhe/Phe¹⁵⁸ effector cells. Importantly, LEX^(Opt) mAbs had significantlyincreased ADCC activity compared to MDX-060 CHO and LEX mAbs,irrespective of the donor genotype. This was assessed by both anincrease in potency and efficacy. Maximal percent lysis for MDX-060Lex^(Opt) was 45% for both experiments, while the EC₅₀ value was 3 to 5times lower than MDX-060 LEX and MDX-060 CHO mAbs, respectively, forFcγRIIIa Val/Phe¹⁵⁸ effector cells and 2 to 3 times lower for theFcγRIIIa Phe/Phe¹⁵⁸ effector cells. These results demonstrate thatremoval of Fuc and Xyl-containing N-glycans from MDX-060 LEX^(Opt) mAbscaused an enhancement in ADCC activity and hence can improve theirtherapeutic potential.

RP-HPLC-Q-TOF MS Analysis of Intact IgG for MDX-060 LEX and MDX-060LEX^(Opt)

Protein A purified IgG's (50 μg) were desalted using the Waters 2695HPLC system fitted with a Poros R1-10 column (2 mm×30 mm; AppliedBiosystems). IgG's were detected and analyzed using a Waters 2487 dualwavelength UV detector (280 nm) and the Waters Q-TOF API US. Separationswere conducted at 0.15 mL/min, 60° C., using 0.05% (v/v) trifluoroaceticacid (TFA; solvent A) and 0.05% (v/v) TFA, 80% (v/v) acetonitrile(solvent B). Sample elution was carried out using a linear increase from30 to 50% B for 5 min, an increase to 80% B for 5 min. The solvent ratioremained at 80% B for an additional 4 min, followed by a wash with 100%B for 1 min and equilibration of the column with 30% B for 15 min priorto the next run.

Q-TOF analysis was conducted in positive ion mode with source anddesolvation temperatures of 100° C. and 300° C., respectively, andcapillary and cone voltages of 3.0 and 60 V, respectively. Data are theresult of combining ≧100 individual scans and deconvolution to theparent mass spectrum using MaxEnt 1.

See also Triguero et al. (2005) Plant Biotechnol. J. 3: 449-457;Takahashi et al. (1998) Anal. Biochem. 255: 183-187; Dillon et al.(2004) J. Chromatogr. A. 1053: 299-305.

FIG. 47 shows intact mass analysis of the MDX-060 LEX mAb compositionsproduced in wild-type L. minor comprising the MDXA01 construct. WhenXylT and FucT expression are not suppressed in L. minor, therecombinantly produced MDX-060 LEX mAb composition comprises at least 7different glycoforms, with the G0XF³ glycoform being the predominatespecies present. Note the absence of a peak representing the G0glycoform.

FIG. 48 shows glycan mass analysis of the heavy chain of the MDX-060 LEXmAb produced in wild-type L. minor comprising the MDXA01 construct. WhenXylT and FucT expression are not suppressed in L. minor, the predominateN-glycan species present is G0XF³, with additional major peaksreflecting the G0X species. Note the minor presence of the G0 glycanspecies.

FIG. 49 shows intact mass analysis of the MDX-060 LEX^(Opt) mAbcompositions produced in transgenic L. minor comprising the MDXA04construct. When XylT and FucT expression are suppressed in L. minor, theintact mAb composition contains only G0 N-glycans. In addition, thecomposition is substantially homogeneous for the G0 glycoform (peak 2),wherein both glycosylation sites are occupied by the G0 N-glycanspecies, with two minor peaks reflecting trace amounts of precursorglycoforms (peak 1, showing mAb having an Fc region wherein the C_(H)2domain of one heavy chain has a G0 glycan species attached to Asn 297,and the C_(H)2 domain of the other heavy chain is unglycosylated; andpeak 3, showing mAb having an Fc region wherein the Asn 297glycosylation site on each of the C_(H)2 domains has a G0 glycan speciesattached, with a third G0 glycan species attached to an additionalglycosylation site within the mAb structure).

FIG. 50 shows glycan mass analysis of the heavy chain of the MDX-060LEX^(Opt) mAb produced in transgenic L. minor comprising the MDXA04construct. When XylT and FucT expression are suppressed in L. minor, theonly readily detectable N-glycan species attached to the Asn 297glycosylation sites of the C_(H)2 domains of the heavy chains is G0.

Discussion

Glyco-optimization of MDX-060 was accomplished by co-expression with anRNAi cassette aimed at silencing the endogenous Lemna FucT and XylTgenes. This simultaneous silencing of both FucT and XylT genes wasachieved using a single RNAi transcript. The absence of Fuc and Xyl onthe LEX^(Opt) mAb was confirmed by MALDI-TOF, NP-HPLC-QTOF MS, andmonosaccharide analysis of N-glycans purified from the MDX-060 LEX^(Opt)mAb. These analyses corroborate the lack of transferase activityobserved in microsomal membranes. Importantly, >95% of the N-glycansreleased from LEX^(Opt) mAbs were of a single structure, GnGn,indicating that this strategy had the added benefit of producing mAbswith a homogeneous N-glycan profile. MDX-060 LEX and LEX^(Opt) mAbs werefound to be indistinguishable with regard to thermal stability andantigen binding compared to MDX-060 CHO. Electrophoretic analysis wasalso found to be similar for all three mAbs. In fact, the onlystructural differences detected were in the mAb N-glycan profiles.

Without being bound by theory, the ability of the MDX-060 LEX^(Opt)lines to produce mAbs with a single major N-glycan species may be basedon the more uniform mAb glycoform distribution found in wild-type Lemna.N-glycans released from mAbs purified from wild-type tobacco (Fujiyamaet al. (2006) J. Biosci. Bioeng. 101:212-218; Elbers et al. (2001) PlantPhysiol. 126:1314-1322; Bakker et al. (2001) Proc. Natl. Acad. Sci.98:2899-2904), alfalfa (Bardor et al. (2003) Plant Biotechnol. J.1:451-462), and moss (Koprivova et al. (2003) Plant Bio. 5:582-591) showthat mAb glycoform heterogeneity in plants with wild-typeN-glycosylation can range from five (alfalfa) (Bardor et al. (2003)Plant Biotechnol. J. 1:451-462) to eight (tobacco) (Fujiyama et al.(2006) J. Biosci. Bioeng. 101:212-218) different major structures.MDX-060 LEX possesses only three major N-glycan structures (GnGn, GnGnXand GnGnXF³). This simple array of N-glycans on mAbs produced bywild-type Lemna may provide a more amenable starting point forglyco-optimization leading to greater homogeneity than that observed inother systems.

Fc-receptor mediated effector cell function has been shown to beimportant for the in vivo activity of many therapeutic mAbs. In thisstudy, the ADCC activity of MDX-060 CHO, MDX-060 LEX, and MDX-060LEX^(Opt) mAbs was compared. Since the FcR expressed on NK cells andmacrophages responsible for ADCC activity is FcγRIIIa, the binding ofthe various mAbs to this receptor was also compared. The resultsdiscussed above show that MDX-060 LEX^(Opt) mAb has an increased bindingaffinity (15-25 fold) and maximal binding (4-5 fold) to FcγRIIIa as wellas enhanced ADCC activity compared to MDX-060 CHO and MDX-060 LEX mAbs.The removal of α-1,6-linked Fuc from various mAbs produced in otherexpression systems has been shown previously to increase FcR binding andenhance ADCC function (Shinkawa et al. (2003) J. Biol. Chem.278:3466-3473; Shields et al. (2002) J. Biol. Chem. 277:26733-26740;Niwa et al. (2004) Clin. Cancer Res. 10:6248-6255). The resultspresented herein suggest that removal of the α-1,3-linked Fuc from theMDX-060 LEX^(Opt) mAbs has the same effect on mAb function as theremoval of α-1,6-linked Fuc.

In this study, two naturally occurring polymorphic isoforms of FcγRIIIaat residue 158⁴¹, Val¹⁵⁸ and Phe¹⁵⁸, were evaluated. MDX-060 LEX^(Opt)shows higher binding affinity to FcγRIIIa-Val¹⁵⁸ compared toFcγRIIIa-Phe¹⁵⁸ as has been observed with other IgG1 mAbs (Shields etal. (2002) J. Biol. Chem. 277:26733-26740). The fact that an increase inbinding with MDX-060 LEX^(Opt) was observed with both isoforms isimportant since differential binding to Val¹⁵⁸ over Phe¹⁵⁸ was found tobe predictive of the clinical and immunological responsiveness ofcertain patient groups receiving anti-CD20 treatment (Cartron et al.(2002) Blood 99:754-758; Weng et al. (2003) J. Clin. Oncol.21:3940-3947; Weng et al. (2004) J. Clin. Oncol. 22:4717-4724). Thisincrease in binding has been hypothesized to result in a higherpercentage of patients responding to treatment that requires Fcfunctionality.

A similar increase in ADCC activity was also observed. In this study,the MDX-060 LEX^(Opt) mAb showed an increase in cell lysis and adecrease in the EC₅₀ value, resulting in an increase in efficacy andpotency when compared to MDX-060 CHO. This corresponds to a 20-foldincrease in activity, determined by taking the maximum percent lysis ofMDX-060 CHO and calculating the concentration of MDX-060 LEX^(Opt) mAbgiving rise to the same percent cell lysis. As with the FcγRIIIa bindingstudy, the increase in ADCC activity was observed with both a homozygousFcγRIIIaPhe/Phe¹⁵⁸ and a heterozygous FcγRIIIa Phe/Val¹⁵⁸ effector celldonor. The results presented here suggest that removal of theα-1,3-linked Fuc from the MDX-060 LEX^(Opt) mAbs has the same effect onmAb function as the removal of α-1,6-linked Fuc.

The robustness of this glyco-optimization strategy has been demonstratedwith multiple independent Lemna plant lines expressing the MDX-060LEX^(Opt) mAb as well as with other mAbs expressed in the Lemnaexpression system (see, for example, Examples 2-4 above). Furthermore,there is no apparent difference in plant phenotype or growth ratecompared with wild-type Lemna plants. Unlike mammalian cell culturesystems where N-glycan heterogeneity can change with culture conditions,growth scale and growth period⁸, the glycan uniformity observed withLEX^(Opt) mAbs has been shown to be consistent under a variety of growthconditions and scales (data not shown).

In conclusion, an RNAi strategy was used to produce a glyco-optimizedanti-CD30 antibody in the Lemna expression system. The resulting mAbconsists of a single, major N-glycan structure, without any evidence ofthe plant-specific Fuc and Xyl residues. In addition, the resultingoptimized mAb has increased ADCC activity and FcγRIIIa binding activitycompared to a CHO-derived mAb. The homogeneous glycosylation profileobtained on mAbs produced in a Lemna expression system having thisFucT+XylT gene knockout strategy makes it is possible to express thesemAbs with increased production consistency.

Example 7 Scale-Up Production of Glycan-Optimized mAbI in Lemna minor

L. minor transgenic line 24 comprising the mAbI04 construct of FIG. 12(providing for suppression of FucT and XylT) was generated in a mannersimilar to that described above. Following its generation, transgenicline 24 was continuously maintained by clonal culture, whereinperiodically a subsample of the plant culture was transferred to freshculture medium for further culturing. This transgenic line was analyzedfor the N-glycosylation pattern of the recombinantly produced mAbIantibody following production scale-up from 1 g tissue up to 300 g (0.3kg) tissue, and further production scale-up to 6.5 kg tissue. Theprocess of scaling production up to 6.5 kg tissue occurred approximately8 months after transgenic line 24 was generated. Results are shown inFIGS. 51A (MALDI-TOF analysis of N-glycans) and 51B (HPLC fluorescenceanalysis of N-glycans).

The glycosylation profile for the mAbI antibody produced by transgenicline 24 comprising the mAbI04 construct remained homogeneous withscale-up in production from 1 g tissue to 0.3 kg tissue, and furtherscale-up in production to 6.5 kg tissue, and thus was characterized bythe presence of a single predominant peak corresponding to the GnGn(i.e., G0) glycan species. Thus, the homogeneity of the glycosylationprofile in transgenic L. minor comprising the mAbI04 construct wasconsistently maintained with an approximately 6,500-fold increase inproduction scale (i.e., from 1 g up to 6.5 kg). Furthermore, thehomogeneity of the glycosylation profile was consistently maintained inthis transgenic line at 8 months following its generation.

These data demonstrate that the homogeneity of the glycosylation profilein transgenic L. minor comprising the mAbI04 construct remainsconsistent with at least a 6,500-fold increase in production scale.Furthermore, the homogeneity of the glycosylation profile in transgenicL. minor comprising the mAbI04 construct is maintained for at least 8months after the transgenic line is generated. The homogeneity of theglycosylation profile would be expected to be maintained with furtherincrease in production scale, and thus, for example, would be expectedto be maintained if production scale was increased by another 4-foldbeyond 6.5 kg (e.g., scale-up from 6.5 kg to 26 kg). The homogeneity ofthe glycosylation profile would also be expected to be maintained withcontinuous clonal culture of the transgenic line well beyond 8 monthsafter generation of the transgenic line.

Example 8 Glycosylation Pattern for Endogenous Glycoproteins in Lemnaminor Lines Transgenic for mAbI04 RNAi Construct

The L40 protease is a representative endogenous glycoprotein produced inL. minor. In order to assess the impact of the mAbI04 RNAi construct(FIG. 12) on glycosylation of endogenous proteins, the L40 protein wasisolated from a L. minor line transgenic for the mAbI04 RNAi constructusing benzamidine affinity chromatography. The N-glycosylation patternfor the isolated L40 protein was analyzed using MALDI-TOF analysis inthe manner described above. Results are shown in FIG. 52.

As can be seen from this analysis, suppression of FucT and XylTexpression using the chimeric RNAi mAbI04 construct results inendogenous glycoproteins having a homogeneous glycosylation patternconsistent with that observed for recombinant glycoproteins. Thus, theheterogeneous N-glycan profile for the L40 glycoprotein isolated from L.minor having the wild-type glycosylation machinery is represented by amixture of N-glycans species having the β1,2-linked xylose residue, orboth the β1,2-linked xylose and core α1,3-linked fucose residuesattached. In contrast, the homogeneous N-glycan profile for L40 isolatedfrom L. minor transgenic for the mAbI04 RNAi construct is represented bya single predominant peak corresponding to the G0 glycan species, and ischaracterized by the absence of N-glycan species having the β1,2-linkedxylose or both the β1,2-linked xylose and core α1,3-linked fucoseresidues attached.

Example 9 Production of Anti-CD20 and Anti-HER2 Monoclonal AntibodyHaving Increased ADCC Activity

IDEC-C2B8 (IDEC Pharmaceuticals Corp., San Diego, Calif.; commerciallyavailable under the tradename Rituxan®, also referred to as rituximab;see U.S. Pat. No. 5,736,137, herein incorporated by reference) is achimeric anti-CD20 monoclonal antibody containing human IgG1 and kappaconstant regions with murine variable regions isolated from a murineanti-CD20 monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood83:435-445). Rituximab is licensed for treatment of relapsed B celllow-grade or follicular non-Hodgkin's lymphoma (NHL). The anti-CD20antibody marketed as rituximab (Rituxan®) is recombinantly produced inCHO cells. The glycosylation pattern of this CHO-expressed anti-CD20antibody reveals a heterogeneous mixture of glycoforms.

A humanized anti-ERBB2 antibody is commercially available under thetradename Herceptin® (Genentech, Inc., San Francisco, Calif.) (see U.S.Pat. No. 6,165,464, herein incorporated by reference). This recombinanthumanized monoclonal antibody has high affinity for p185HER2. Earlyclinical trials with patients having extensive metastatic breastcarcinomas demonstrate the ability of this monoclonal antibody toinhibit growth of breast cancer cells that overexpress HER2 (Baselga etal. (1996) J. Clin. Oncol. 14(3):737-744).

A rituximab-sequence antibody and Herceptin® anti-ERBB2-sequenceantibody are recombinantly produced in Lemna having a wild-typeglycosylation pattern, using the mAbI01 construct described above, andin Lemna that is genetically modified to suppress expression of bothFucT and XylT, using the mAbI04 chimeric RNAi construct, with therituximab or anti-ERBB2 heavy and light chain coding sequences replacingthose for the mAbI in each of these constructs. The sequences encodingthe heavy and light chains are optionally both codon-optimized withLemna-preferred codons. The secreted rituximab-sequence mAb (i.e.,having the amino acid sequence of the rituximab antibody) andanti-ERBB2-sequence mAb (i.e., having the amino acid sequence of theHerceptin® anti-ERBB2 mAb) are analyzed for glycosylation pattern asdescribed above in Example 1.

The glycosylation profile for intact rituximab-sequence mAb oranti-ERBB2-sequence mAb produced in the wild-type Lemna comprising themAbI01-like construct shows a heterogeneous profile with numerous peakscorresponding to multiple glycoforms. In contrast, the glycoyslationprofile for intact rituximab-sequence mAb or intact anti-ERBB2-sequencemAb produced in transgenic Lemna comprising the mAbI04-like constructshows a substantially homogeneous glycoprotein composition, with threemajor glycoform peaks, the largest of which corresponds to the G0glycoform, and two very minor peaks corresponding to trace amounts ofprecursor glycoforms, wherein xylose and fucose residues are notattached.

The rituximab-sequence and anti-ERBB2-sequence monoclonal antibodycompositions having a glycosylation profile that is substantiallyhomogeneous for the G0 glycoform are tested for ADCC activity and Fcγreceptor IIIa (Fcγ RIIIa) binding on freshly-isolated human NK cells.The rituximab-sequence and anti-ERBB2-sequence mAbs produced from L.minor lines engineered with the mAbI04-like construct exhibit theexpected enhanced binding in view of the lack of any fucose residues,relative to the binding observed for the rituximab-sequence andanti-ERBB2-sequence mAbs produced from L. minor lines having thewild-type glycosylation machinery (i.e., no silencing of FucT or XylT).Furthermore, binding affinity is at least strong as for thecorresponding mAb produced in CHO cell lines.

ADCC activity of the substantially homogeneous G0 glycoform ofrituximab-sequence mAb and of anti-ERBB2-sequence mAb produced in the L.minor line engineered with the mAbI04-like construct is assayed usingpurified human peripheral blood mononuclear cells as effector cells(see, for example, Shinkawa et al. (2003) J. Biol. Chem.278(5):3466-3473; herein incorporated by reference in its entirety).Activity of the G0 glycoform rituximab-sequence and anti-ERBB2-sequencemAb compositions is improved 50-1000 fold over that exhibited by therespective rituximab-sequence mAb or anti-ERBB2-sequence mAb produced inthe L. minor line having the wild-type glycosylation machinery orproduced in CHO cells.

CDC activity of the substantially homogeneous G0 glycoform ofrituximab-sequence mAb produced in the L. minor line engineered with themAbI04-like RNAi construct is assayed using standard assays known in theart (see, for example, the complement activation assays described inU.S. Patent Application Publication No. 2004/0167319, hereinincorporated by reference in its entirety), and compared to thatobserved for rituximab. Non-relevant antibody serves as the negativecontrol. In one such assay, CDC activity of the various antibodiesagainst target Daudi cells is measured by assessing elevated membranepermeability using a propidium iodide (PI) exclusion assay, with serumfrom healthy volunteers serving as a complement source. Serum forcomplement lysis is prepared by drawing blood from healthy volunteersinto autosep gel and clot activator vacutainer tubes (BD Biosciences,Rutherford, N.J.), which are held at room temperature for 30-60 minutesand then centrifuged at 3000 rpm for 5 minutes. Serum is harvested andstored at −80° C.

Briefly, for this CDC activity assay, Daudi cells are washed andresuspended in RPMI-1% BSA at 1×10⁶/ml. Various concentrations of thesubstantially homogeneous G0 glycoform of rituximab-sequence mAb,rituximab, and negative control mAb are added to the Daudi cells andallowed to bind for 10-15 minutes at room temperature. Thereafter, serumas a source of complement is added to a final concentration of 20% (v/v)and the mixtures are incubated for 45 min at 37° C. The cells are thenkept at 4° C. until analysis. Each sample (150 μl) is then added to 10μl of PI solution (10 μg/ml in PBS) in a FACS tube. The mixture isassessed immediately for cell lysis (number of PI-positive cells) by aFACScalibur flow cytometer and analysed using CellQuest pro software (BDBiosciences, Mountain View, Calif.). At least 5000 events are collectedfor analysis with cell debris excluded by adjustment of the forwardsideward scatter (FCS) threshold.

CDC activity of the substantially homogeneous G0 glycoformrituximab-sequence mAb is decreased relative to that exhibited byrituximab.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1. A duckweed plant or duckweed plant cell or nodule that expressesglycoproteins having an altered N-glycosylation pattern, wherein saidaltered N-glycosylation pattern is characterized by a reduction in theattachment of α1,3-fucose residues, β1,2-xylose residues, or bothα1,3-fucose residues and β1,2-xylose residues to N-glycans of saidglycoproteins when compared to the N-glycosylation pattern for saidglycoproteins when expressed in a control duckweed plant or duckweedplant cell or nodule, wherein said reduction in said duckweed plant orduckweed plant cell or nodule results from inhibiting expression in saidduckweed plant or duckweed plant cell or nodule of at least oneα1,3-fucosyltransferase (FucT) comprising an amino acid sequence havingat least 90% sequence identity to the sequence set forth in SEQ ID NO:3and, at least one β1,2-xylosyltransferase (XylT) comprising an aminoacid sequence having at least 90% sequence identity to the sequence setforth in SEQ ID NO:6 or SEQ ID NO:21, wherein said control duckweedplant or duckweed plant cell or nodule has not been genetically modifiedto inhibit expression of said FucT or said XylT in said control duckweedplant or duckweed plant cell or nodule.
 2. The duckweed plant orduckweed plant cell or nodule of claim 1, wherein said N-glycans aredevoid of said fucose residues, devoid of said xylose residues, ordevoid of said fucose and xylose residues.
 3. The duckweed plant orduckweed plant cell or nodule of claim 1, wherein said duckweed plant orduckweed plant cell or nodule comprises a polynucleotide encoding amammalian glycoprotein of interest.
 4. The duckweed plant or duckweedplant cell or nodule of claim 3 , wherein said glycoprotein is amonoclonal antibody of interest.
 5. The duckweed plant or duckweed plantcell or nodule of claim 4, wherein said monoclonal antibody hasincreased binding affinity for an FcγRIII, increased antibody-dependentcellular cytotoxicity (ADCC), decreased complement-dependentcytotoxicity (CDC) activity, or any combination thereof, as a result ofsaid altered N-glycosylation pattern of said monoclonal antibody.
 6. Amethod for altering the N-glycosylation pattern of a heterologouspolypeptide produced in a duckweed plant or duckweed plant cell ornodule, said method comprising: (a) inhibiting expression of at leastone α1,3-fucosyltransferase (FucT) in said a duckweed plant or duckweedplant cell or nodule, wherein said FucT comprises an amino acid sequencehaving at least 90% sequence identity to the sequence set forth in SEQID NO:3; and (b) culturing said duckweed plant or duckweed plant cell ornodule under conditions suitable for expression of said heterologouspolypeptide.
 7. The method of claim 5, wherein the expression of saidFucT is inhibited by a method selected from the group consisting of: (a)introducing a polynucleotide into said duckweed plant or duckweed plantcell or nodule, wherein said polynucleotide inhibits expression of saidFucT in said a duckweed plant or duckweed plant cell or nodule; (b)eliminating a gene in said duckweed plant or duckweed plant cell ornodule, wherein said gene encodes said FucT; and (c) mutating a gene insaid duckweed plant or duckweed plant cell or nodule, wherein said geneencodes said FucT.
 8. The method of claim 7, comprising introducing intosaid duckweed plant or duckweed plant cell or nodule a nucleotideconstruct comprising a first nucleotide sequence that is capable ofinhibiting expression of said α1,3-fucosyltransferase (FucT) in saidduckweed plant or duckweed plant cell or nodule, wherein said firstnucleotide sequence is operably linked to a promoter that is functionalin a plant cell.
 9. The method of claim 8, wherein said first nucleotidesequence comprises a sequence selected from the group consisting of: (a)the nucleotide sequence set forth in SEQ ID NO:1 or a complementthereof; (b) the nucleotide sequence set forth in SEQ ID NO:2 or acomplement thereof; (c) a nucleotide sequence having at least 90%sequence identity to the sequence of preceding item (a) or (b); and (d)a fragment of the nucleotide sequence of any one of preceding items (a)through (c), wherein said fragment comprises at least 75 contiguousnucleotides of said nucleotide sequence.
 10. The method of claim 8,wherein said first nucleotide sequence comprises in the 5′-to-3′orientation and operably linked: (a) a FucT forward fragment, said FucTforward fragment comprising about 500 to about 800 contiguousnucleotides having at least 90% sequence identity to a nucleotidesequence of about 500 to about 800 contiguous nucleotides of SEQ ID NO:1or SEQ ID NO:2; (b) a spacer sequence comprising about 200 to about 700nucleotides; and (c) a FucT reverse fragment, said FucT reverse fragmenthaving sufficient length and sufficient complementarity to said FucTforward fragment such that said first nucleotide sequence is transcribedas an RNA molecule capable of forming a hairpin RNA structure.
 11. Themethod of claim 10, wherein said FucT reverse fragment comprises thecomplement of said FucT forward fragment or a sequence having at least90% sequence identity to the complement of said FucT forward fragment.12. The method of claim 10, wherein said FucT forward fragment comprisesnucleotides (nt) 255-985 of SEQ ID NO:1 and said spacer sequencecomprises an intron.
 13. The method of claim 6, further comprisinginhibiting expression of at least one β1,2-xylosyltransferase (XylT) insaid duckweed plant or duckweed plant cell or nodule, wherein said XyltTcomprises an amino acid sequence having at least 90% sequence identityto the sequence set forth in SEQ ID NO:6 or SEQ ID NO:21.
 14. The methodof claim 13, wherein the expression of said XylT is inhibited by amethod selected from the group consisting of: (a) introducing apolynucleotide into said duckweed plant or duckweed plant cell ornodule, wherein said polynucleotide inhibits expression of said XylT insaid a duckweed plant or duckweed plant cell or nodule; (b) eliminatinga gene in said duckweed plant or duckweed plant cell or nodule, whereinsaid gene encodes said XylT; and (c) mutating a gene in said duckweedplant or duckweed plant cell or nodule, wherein said gene encodes saidXylT.
 15. The method of claim 14, comprising introducing into saidduckweed plant or duckweed plant cell or nodule a nucleotide constructcomprising a nucleotide sequence that is capable of inhibitingexpression of said XylT in said duckweed plant or duckweed plant cell ornodule, wherein said nucleotide sequence that is capable of inhibitingexpression of said XylT is operably linked to a promoter that isfunctional in a plant cell.
 16. The method of claim 15, wherein saidnucleotide sequence that is capable of inhibiting expression of saidXylT comprises a sequence selected from the group consisting of: (a) thenucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:19, or acomplement thereof; (b) the nucleotide sequence set forth in SEQ IDNO:5, SEQ ID NO:20, or a complement thereof; (c) a nucleotide sequencehaving at least 90% sequence identity to the sequence of preceding item(a) or (b); and (d) a fragment of the nucleotide sequence of any one ofpreceding items (a) through (c), wherein said fragment comprises atleast 75 contiguous nucleotides of said nucleotide sequence.
 17. Themethod of claim 15, wherein said nucleotide sequence that is capable ofinhibiting expression of said XylT comprises in the 5′-to-3′orientationand operably linked: (a) a XylT forward fragment, said XylT forwardfragment comprising about 500 to about 800 contiguous nucleotides havingat least 90% sequence identity to a nucleotide sequence of about 500 toabout 800 contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:19, or SEQ ID NO:20; (b) a spacer sequence comprising about 200 toabout 700 nucleotides; and (c) a XylT reverse fragment, said XylTreverse fragment having sufficient length and sufficient complementarityto said XylT forward fragment such that said nucleotide sequence that iscapable of inhibiting expression of said XylT is transcribed as an RNAmolecule capable of forming a hairpin RNA structure.
 18. The method ofclaim 17, wherein said XylT reverse fragment comprises the complement ofsaid XylT forward fragment or a sequence having at least 90% sequenceidentity to the complement of said XylT forward fragment.
 19. The methodof claim 17, wherein: (a) said XylT forward fragment comprisesnucleotides (nt) 318-1052 of SEQ ID NO:4 and wherein said spacersequence is an intron; or (b) said XylT forward fragment comprises nt1-734 of SEQ ID NO:19 and said spacer sequence is an intron.
 20. Themethod of claim 13, wherein a nucleotide construct comprising a fusionpolynucleotide that is capable inhibiting expression of said FucT andsaid XylT in said duckweed plant or duckweed plant cell or nodule isintroduced into said duckweed plant or duckweed plant cell or nodule,wherein said fusion polynucleotide is operably linked to a promoter thatis functional in a plant cell.
 21. The method of claim 20, wherein saidfusion polynucleotide comprises in the 5′-to-3′ orientation and operablylinked: (a) a chimeric forward fragment, said chimeric forward fragmentcomprising in either order: (i) a first fragment comprising about 500 toabout 650 contiguous nucleotides having at least 90% sequence identityto a nucleotide sequence of about 500 to about 650 contiguousnucleotides of SEQ ID NO:1 or SEQ ID NO:2; and (ii) a second fragmentcomprising about 500 to about 650 contiguous nucleotides having at least90% sequence identity to a nucleotide sequence of about 500 to about 650contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQID NO:20; (b) a spacer sequence comprising about 200 to about 700nucleotides; (c) a reverse fragment, said reverse fragment havingsufficient length and sufficient complementarity to said chimericforward fragment such that said fusion polynucleotide is transcribed asan RNA molecule capable of forming a hairpin RNA structure.
 22. Themethod of claim 21, wherein said first fragment comprises about 500 toabout 650 contiguous nucleotides of SEQ ID NO:1; and said secondfragment comprises about 500 to about 650 contiguous nucleotides of SEQID NO:4 or SEQ ID NO:19.
 23. The method of claim 22, wherein saidreverse fragment comprises the complement of said chimeric forwardfragment or a sequence having at least 90% sequence identity to thecomplement of said chimeric forward fragment.
 24. The method of claim21, wherein: (a) said chimeric forward fragment comprises nucleotides(nt) 254-855 of SEQ ID NO:1 and nt 318-943 of SEQ ID NO:4, and whereinsaid spacer sequence is an intron; or (b) said chimeric forward fragmentcomprises nucleotides (nt) 254-855 of SEQ ID NO:1 and nt 1-626 of SEQ IDNO:19, and wherein said spacer sequence is an intron.
 25. A method foraltering the N-glycosylation pattern of a heterologous polypeptideproduced in a duckweed plant or duckweed plant cell or nodule, saidmethod comprising: (a) inhibiting expression of at least one aβ1,2-xylosyltransferase (XylT) in said duckweed plant or duckweed plantcell or nodule, wherein said XyltT comprises an amino acid sequencehaving at least 90% sequence identity to the sequence set forth in SEQID NO:6 or SEQ ID NO:21; and (b) culturing said duckweed plant orduckweed plant cell or nodule under conditions suitable for expressionof said heterologous polypeptide.
 26. The method of claim 25, whereinthe expression of said XylT is inhibited by a method selected from thegroup consisting of: (a) introducing a polynucleotide into said duckweedplant or duckweed plant cell or nodule, wherein said polynucleotideinhibits expression of said XylT in said a duckweed plant or duckweedplant cell or nodule; (b) eliminating a gene in said duckweed plant orduckweed plant cell or nodule, wherein said gene encodes said XylT; and(c) mutating a gene in said duckweed plant or duckweed plant cell ornodule, wherein said gene encodes said XylT.
 27. The method of claim 26,comprising introducing into said duckweed plant or duckweed plant cellor nodule a nucleotide construct comprising a nucleotide sequence thatis capable of inhibiting expression of said XylT in said duckweed plantor duckweed plant cell or nodule, wherein said nucleotide sequence thatis capable of inhibiting expression of said XylT is operably linked to apromoter that is functional in a plant cell.
 28. The method of claim 27,wherein said nucleotide sequence that is capable of inhibitingexpression of said XylT comprises a sequence selected from the groupconsisting of: (a) the nucleotide sequence set forth in SEQ ID NO:4, SEQID NO:19, or a complement thereof; (b) the nucleotide sequence set forthin SEQ ID NO:5, SEQ ID NO:20, or a complement thereof; (c) a nucleotidesequence having at least 90% sequence identity to the sequence ofpreceding item (a) or (b); and (d) a fragment of the nucleotide sequenceof any one of preceding items (a) through (c), wherein said fragmentcomprises at least 75 contiguous nucleotides of said nucleotidesequence.
 29. The method of claim 27, wherein said nucleotide sequencethat is capable of inhibiting expression of said XylT comprises in the5′-to-3′orientation and operably linked: (a) a XylT forward fragment,said XylT forward fragment comprising about 500 to about 800 contiguousnucleotides having at least 90% sequence identity to a nucleotidesequence of about 500 to about 800 contiguous nucleotides of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:21; (b) a spacer sequencecomprising about 200 to about 700 nucleotides; and (c) a XylT reversefragment, said XylT reverse fragment having sufficient length andsufficient complementarity to said XylT forward fragment such that saidnucleotide sequence that is capable of inhibiting expression of saidXylT is transcribed as an RNA molecule capable of forming a hairpin RNAstructure.
 30. The method of claim 29, wherein said XylT reversefragment comprises the complement of said XylT forward fragment or asequence having at least 90% sequence identity to the complement of saidXylT forward fragment.
 31. The method of claim 29, wherein: (a) saidXylT forward fragment comprises nucleotides (nt) 318-1052 of SEQ ID NO:4and wherein said spacer sequence is an intron; or (b) said XylT forwardfragment comprises nt 1-734 of SEQ ID NO:19 and wherein said spacersequence is an intron.
 32. The method of claim 6, wherein saidheterologous polypeptide is a mammalian polypeptide.
 33. The method ofclaim 32, wherein said mammalian polypeptide is selected from the groupconsisting of an interferon, erythropoietin (EPO), tissue plasminogenactivator (tPA), plasminogen, blood coagulation factors,granulocyte-macrophage colony stimulating factor (GM-CSF), andtherapeutic immunoglobulins.
 34. A nucleotide construct comprising afirst nucleotide sequence that is capable of inhibiting expression of anα1,3-fucosyltransferase (FucT) in a duckweed plant, and a secondnucleotide sequence that is capable of inhibiting expression of aβ1,2-xylosyltransferase (XylT) in a duckweed plant, wherein said firstnucleotide sequence and said second nucleotide sequence are operablylinked to at least one promoter that is functional in a plant cell;wherein said FucT is a polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence setforth in SEQ ID NO:3; and (b) an amino acid sequence having at least 90%sequence identity to the amino acid sequence set forth in SEQ ID NO:3;and wherein said XylT is a polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence setforth in SEQ ID NO:6 or SEQ ID NO:21; and (b) an amino acid sequencehaving at least 90% sequence identity to the amino acid sequence setforth in SEQ ID NO:6 or SEQ ID NO:21.
 35. The nucleotide construct ofclaim 34, wherein said first nucleotide sequence is operably linked to afirst promoter, and wherein said second nucleotide sequence is operablylinked to a second promoter.
 36. The nucleotide construct of claim 34,wherein said first nucleotide sequence comprises a sequence selectedfrom the group consisting of: (a) the nucleotide sequence set forth inSEQ ID NO:1 or a complement thereof; (b) the nucleotide sequence setforth in SEQ ID NO:2 or a complement thereof; (c) a nucleotide sequencehaving at least 90% sequence identity to the sequence of preceding item(a) or (b); and (d) a fragment of the nucleotide sequence of any one ofpreceding items (a) through (c), wherein said fragment comprises atleast 75 contiguous nucleotides of said nucleotide sequence.
 37. Thenucleotide construct of claim 34, wherein said first nucleotide sequencecomprises in the 5′-to-3′ orientation and operably linked: (a) a FucTforward fragment, said FucT forward fragment comprising about 500 toabout 800 contiguous nucleotides having at least 90% sequence identityto a nucleotide sequence of about 500 to about 800 contiguousnucleotides of SEQ ID NO:1 or SEQ ID NO:2; (b) a spacer sequencecomprising about 200 to about 700 nucleotides; and (c) a FucT reversefragment, said FucT reverse fragment having sufficient length andsufficient complementarity to said FucT forward fragment such that saidfirst nucleotide sequence is transcribed as an RNA molecule capable offorming a hairpin RNA structure.
 38. The nucleotide construct of claim37, wherein said FucT reverse fragment comprises the complement of saidFucT forward fragment or a sequence having at least 90% sequenceidentity to the complement of said FucT forward fragment.
 39. Thenucleotide construct of claim 37, wherein said FucT forward fragmentcomprises nucleotides (nt) 255-985 of SEQ ID NO:1.
 40. The nucleotideconstruct of claim 37, wherein said spacer sequence of said firstnucleotide sequence comprises an intron.
 41. The nucleotide construct ofclaim 34, wherein said second nucleotide sequence comprises a sequenceselected from the group consisting of: (a) the nucleotide sequence setforth in SEQ ID NO:4 or SEQ ID NO:19, or a complement thereof; (b) thenucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:20, or acomplement thereof; (c) a nucleotide sequence having at least 90%sequence identity to the sequence of preceding item (a) or (b); and (d)a fragment of the nucleotide sequence of any one of preceding items (a)through (c), wherein said fragment comprises at least 75 contiguousnucleotides of said nucleotide sequence.
 42. The nucleotide construct ofclaim 34, wherein said second nucleotide sequence comprises in the5′-to-3′ orientation and operably linked: (a) a XylT forward fragment,said XylT forward fragment comprising about 500 to about 800 contiguousnucleotides having at least 90% sequence identity to a nucleotidesequence of about 500 to about 800 contiguous nucleotides of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:2; (b) a spacer sequencecomprising about 200 to about 700 nucleotides; and (c) a XylT reversefragment, said XylT reverse fragment having sufficient length andsufficient complementarity to said XylT forward fragment such that saidsecond nucleotide sequence is transcribed as an RNA molecule capable offorming a hairpin RNA structure.
 43. The nucleotide construct of claim42, wherein said XylT reverse fragment comprises the complement of saidXylT forward fragment or a sequence having at least 90% sequenceidentity to the complement of said XylT forward fragment.
 44. Thenucleotide construct of claim 42, wherein said XylT forward fragmentcomprises nucleotides (nt) 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ IDNO:19.
 45. The nucleotide construct of claim 42, wherein said spacersequence within said second nucleotide sequence comprises an intron. 46.A nucleotide construct comprising a fusion polynucleotide that iscapable of inhibiting expression of an α1,3-fucosyltransferase (FucT)and a β1,2-xylosyltransferase (XylT) in a duckweed plant, wherein saidfusion polynucleotide is operably linked to a promoter that isfunctional in a plant cell, and wherein said fusion polynucleotidecomprises in the 5′-to-3′ orientation and operably linked: (a) achimeric forward fragment, said chimeric forward fragment comprising ineither order: (i) a first fragment comprising about 500 to about 650contiguous nucleotides having at least 90% sequence identity to anucleotide sequence of about 500 to about 650 contiguous nucleotides ofSEQ ID NO:1 or SEQ ID NO:2; and (ii) a second fragment comprising about500 to about 650 contiguous nucleotides having at least 90% sequenceidentity to a nucleotide sequence of about 500 to about 650 contiguousnucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:2;(b) a spacer sequence comprising about 200 to about 700 nucleotides; and(c) a reverse fragment, said reverse fragment having sufficient lengthand sufficient complementarity to said chimeric forward fragment suchthat said fusion polynucleotide is transcribed as an RNA moleculecapable of forming a hairpin RNA structure.
 47. The nucleotide constructof claim 46, wherein said reverse fragment comprises the complement ofsaid chimeric forward fragment or a sequence having at least 90%sequence identity to the complement of said chimeric forward fragment.48. The nucleotide construct of claim 46, wherein said spacer sequencecomprises an intron.
 49. The nucleotide construct of claim 46, whereinsaid first fragment comprises about 500 to about 650 contiguousnucleotides of SEQ ID NO:1; and said second fragment comprises about 500to about 650 contiguous nucleotides of SEQ ID NO:4 or SEQ ID NO:19. 50.The nucleotide construct of claim 49, wherein said reverse fragmentcomprises the complement of said chimeric forward fragment or a sequencehaving at least 90% sequence identity to the complement of said chimericforward fragment.
 51. The nucleotide construct of claim 49, wherein saidchimeric forward fragment comprises: (a) nucleotides (nt) 254-855 of SEQID NO:1 and nt 318-943 of SEQ ID NO:4; or (b) nucleotides (nt) 254-855of SEQ ID NO:1 and nt 1-626 of SEQ ID NO:19.
 52. The nucleotideconstruct of claim 49, wherein said spacer sequence comprises an intron.53. A nucleotide construct comprising a first polynucleotide sequencethat is capable of inhibiting expression of an α1,3-fucosyltransferase(FucT) in a duckweed plant, wherein said first polynucleotide sequencecomprises in the 5′-to-3′ orientation and operably linked: (a) a FucTforward fragment, said FucT forward fragment comprising about 500 toabout 800 contiguous nucleotides having at least 90% sequence identityto a nucleotide sequence of about 500 to about 800 contiguousnucleotides of SEQ ID NO:1 or SEQ ID NO:2; (b) a spacer sequencecomprising about 200 to about 700 nucleotides; and (c) a FucT reversefragment, said FucT reverse fragment having sufficient length andsufficient complementarity to said FucT forward fragment such that saidfirst polynucleotide sequence is transcribed as an RNA molecule capableof forming a hairpin RNA structure; wherein said first polynucleotidesequence is operably linked to a promoter that is functional in a plantcell.
 54. The nucleotide construct of claim 53, wherein said FucTreverse fragment comprises the complement of said FucT forward fragmentor a sequence having at least 90% sequence identity to the complement ofsaid FucT forward fragment.
 55. The nucleotide construct of claim 53,wherein said spacer sequence of said first polynucleotide comprises anintron.
 56. The nucleotide construct of claim 53, wherein said FucTforward fragment comprises about 500 to about 800 contiguous nucleotidesof SEQ ID NO:1 or SEQ ID NO:2.
 57. The nucleotide construct of claim 56,wherein said FucT reverse fragment comprises the complement of said FucTforward fragment or a sequence having at least 90% sequence identity tothe complement of said FucT forward fragment.
 58. The nucleotideconstruct of claim 56, wherein said FucT forward fragment comprisesnucleotides (nt) 255-985 of SEQ ID NO:1.
 59. The nucleotide construct ofclaim 56, wherein said spacer sequence of said first polynucleotidesequence comprises an intron.
 60. A nucleotide construct comprising afirst polynucleotide sequence that is capable of inhibiting expressionof a β1,2-xylosyltransferase (XylT) in a duckweed plant, wherein saidfirst polynucleotide sequence comprises in the 5′-to-3′ orientation andoperably linked: (a) a XylT forward fragment, said XylT forward fragmentcomprising about 500 to about 800 contiguous nucleotides having at least90% sequence identity to a nucleotide sequence of about 500 to about 800contiguous nucleotides of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQID NO:20; (b) a spacer sequence comprising about 200 to about 700nucleotides; and (c) a XylT reverse fragment, said XylT reverse fragmenthaving sufficient length and sufficient complementarity to said XylTforward fragment such that said first polynucleotide sequence istranscribed as an RNA molecule capable of forming a hairpin RNAstructure; wherein said first polynucleotide sequence is operably linkedto a promoter that is functional in a plant cell.
 61. The nucleotideconstruct of claim 60, wherein said XylT reverse fragment comprises thecomplement of said XylT forward fragment or a sequence having at least90% sequence identity to the complement of said XylT forward fragment.62. The nucleotide construct of claim 60, wherein said spacer sequencewithin said first polynucleotide sequence comprises an intron.
 63. Thenucleotide construct of claim 60, wherein said XylT forward fragmentcomprises about 500 to about 800 contiguous nucleotides of SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:20.
 64. The nucleotide constructof claim 63, wherein said XylT reverse fragment comprises the complementof said XylT forward fragment or a sequence having at least 90% sequenceidentity to the complement of said XylT forward fragment.
 65. Thenucleotide construct of claim 63, wherein said XylT forward fragmentcomprises nucleotides nt 318-1052 of SEQ ID NO:4 or nt 1-734 of SEQ IDNO:19.
 66. The nucleotide construct of claim 63, wherein said spacersequence within said first polynucleotide sequence comprises an intron.67. The nucleotide construct of claim 46, further comprising at leastone polynucleotide encoding a polypeptide of interest, wherein saidpolynucleotide encoding said polypeptide of interest is operably linkedto a promoter that is functional in a plant cell.
 68. The nucleotideconstruct of claim 67, wherein said polypeptide of interest is amammalian polypeptide.
 69. The nucleotide construct of claim 68, whereinsaid mammalian polypeptide is selected from the group consisting of aninterferon, erythropoietin (EPO), tissue plasminogen activator (tPA),plasminogen, blood coagulation factors, granulocyte-macrophage colonystimulating factor (GM-CSF), and therapeutic immunoglobulins.
 70. Avector comprising the nucleotide construct according to claim
 46. 71. Aduckweed plant or duckweed plant cell or nodule comprising thenucleotide construct according to claim 46 stably integrated into itsgenome.
 72. The duckweed plant or duckweed plant cell or nodule of claim71, wherein said duckweed is from a genus selected from the groupconsisting of the genus Spirodela, genus Wolffia, genus Wolfiella, genusLandoltia, and genus Lemna.
 73. The duckweed plant or plant cell ofclaim 72, wherein said duckweed is a member of a species selected fromthe group consisting of Lemna minor, Lemna miniscula, Lemnaaequinoctialis, and Lemna gibba.
 74. A method for stably transforming aduckweed plant to express glycoproteins having an alteredN-glycosylation pattern, said method comprising introducing into saidduckweed plant the nucleotide construct according to claim
 46. 75. Themethod of claim 74, wherein said duckweed plant expresses at least oneheterologous polypeptide of interest.
 76. A method for stablytransforming a duckweed plant to express a heterologous polypeptide ofinterest having an altered N-glycosylation pattern, said methodcomprising introducing into said duckweed plant the nucleotide constructaccording to claim
 67. 77. The method of claim 76, wherein theN-glycosylation pattern is characterized by a reduction in theattachment of α1,3-fucose residues to N-glycans attached to saidheterologous polypeptide, a reduction in the attachment of β1,2-xyloseresidues to N-glycans attached to said heterologous polypeptide, or botha reduction in the attachment of α1,3-fucose residues and β1,2-xyloseresidues to N-glycans attached to said heterologous polypeptide_whencompared to the N-glycosylation pattern of said heterologous polypeptidewhen expressed in a control duckweed plant that has not been geneticallymodified to inhibit expression of said FucT and said XylT in saidcontrol duckweed plant.
 78. A method for producing a heterologousmammalian glycoprotein in a duckweed plant, wherein said heterologousmammalian glycoprotein has a reduction in the attachment of α1,3-fucoseand β1,2-xylose residues to N-glycans of said glycoprotein when producedin said duckweed plant when compared to N-glycans of said glycoproteinwhen produced in a control duckweed plant that has not been geneticallymodified to inhibit expression of α1,3-fucosyltransferase (FucT) andβ1,2-xylosyltransferase (XylT) in said control duckweed plant, saidmethod comprising: (a) introducing into said duckweed plant anexpression cassette comprising a sequence encoding a mammalianpolypeptide that is post-translationally processed as said glycoprotein,and a polynucleotide comprising the nucleotide construct of claim 46;and (b) culturing said duckweed plant under conditions suitable forexpression of said glycoprotein.
 79. The method of claim 78, whereinsaid mammalian polypeptide is selected from the group consisting of aninterferon, erythropoietin (EPO), tissue plasminogen activator (tPA),plasminogen, blood coagulation factors, granulocyte-macrophage colonystimulating factor (GM-CSF), and therapeutic immunoglobulins.
 80. Amethod for reducing heterogeneity of the N-glycosylation profile of aglycoprotein produced in a duckweed plant, said method comprisingintroducing into said duckweed plant the nucleotide construct accordingto claim 46, and culturing said duckweed plant under conditions suitablefor expression of said glycoprotein.
 81. The method of claim 80, whereinsaid glycoprotein is an endogenous glycoprotein.
 82. The method of claim80, wherein said glycoprotein is a heterologous glycoprotein.
 83. Themethod according to claim 80, wherein the reduced heterogeneity of saidN-glycosylation profile is maintained with scale-up in production ofsaid duckweed plant, wherein production scale is increased by at least6,500-fold.
 84. The method according to claim 80, wherein the reducedheterogeneity of said N-glycosylation profile is maintained withcontinuous clonal culture of said duckweed plant.
 85. The method ofclaim 84, wherein the reduced heterogeneity of said N-glycosylationprofile is maintained with continuous clonal culture of said duckweedplant for at least 8 months.
 86. An isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2; (b) thenucleotide sequence set forth in SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:19, or SEQ ID NO:20; (c) a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:3, SEQ IDNO:6; or SEQ ID NO:21; (d) a nucleotide sequence comprising at least 90%sequence identity to the sequence set forth in SEQ ID NO:1 or SEQ IDNO:2, wherein said polynucleotide encodes a polypeptide havingα1,3-fucosyltransferase (FucT) activity; (e) a nucleotide sequencecomprising at least 90% sequence identity to the sequence set forth inSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:2, wherein saidpolynucleotide encodes a polypeptide having β1,2-xylosyltransferase(XylT) activity; (f) a nucleotide sequence comprising at least 50contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:19, or SEQ ID NO:20, or a complement thereof; (g) anucleotide sequence comprising at least 50contiguous nucleotides of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, or SEQ IDNO:20 and a complement thereof; (h) a nucleotide sequence encoding anamino acid sequence having at least 95% sequence identity to thesequence set forth in SEQ ID NO:3, wherein said polynucleotide encodes apolypeptide having FucT activity; (i) a nucleotide sequence encoding anamino acid sequence having at least 95% sequence identity to thesequence set forth in SEQ ID NO:6 or SEQ ID NO:2, wherein saidpolynucleotide encodes a polypeptide having XylT activity; and (j) thecomplement of the nucleotide sequence of any one of preceding items (a)through (i).